Microscopy 2009 Wk14 TEMd

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Microscopy Week 14Microscopy Week 14The Transmission Electron The Transmission Electron

MicroscopeMicroscope12/16/200912/16/2009

Electron MicroscopyElectron Microscopy Principles and techniques for Biologists (eBook)Principles and techniques for Biologists (eBook)2nd edition2nd editionBy L JBy L J. Bozzola. BozzolaL. RussellL. Russellhttp://http://www.netLibrary.com/urlapi.asp?actionwww.netLibrary.com/urlapi.asp?action==summary&vsummary&v=1&bookid=25575=1&bookid=25575

http://www.netlibrary.com/urlapi.asp?action=summary&v=1&bookid=25575





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Final presentationFinal presentation Final project presentation: 1/6/2010 (Wed) Final project presentation: 1/6/2010 (Wed)

2:10 PM2:10 PM1.1. any topic related to microscopy, review, any topic related to microscopy, review,

new concept, instrument, improvement, new concept, instrument, improvement, application,…application,…

2.2. 15 minutes per person15 minutes per person3.3. Paper report, (summery the reference you Paper report, (summery the reference you

use and make a commend about any use and make a commend about any uniqueness, advantages and uniqueness, advantages and disadvantages) disadvantages)

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ContentsContents Basic Systems Making Up a Transmission Electron Basic Systems Making Up a Transmission Electron

MicroscopeMicroscope Illuminating SystemIlluminating System Specimen Manipulation SystemSpecimen Manipulation System Imaging SystemImaging System Vacuum SystemVacuum System

VISIBLE LIGHT, ELECTRONS, AND LENSESVISIBLE LIGHT, ELECTRONS, AND LENSES Electromagnetic Radiation and the Diffraction PhenomenonElectromagnetic Radiation and the Diffraction Phenomenon Effect of Diffraction on ResolutionEffect of Diffraction on Resolution Electrons, Waves, and ResolutionElectrons, Waves, and Resolution General Design of LensesGeneral Design of Lenses Design of Electromagnetic LensesDesign of Electromagnetic Lenses

DEFECTS IN LENSESDEFECTS IN LENSES

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ContentsContents MAGNIFICATION MAGNIFICATION DESIGN OF THE TRANSMISSION ELECTRON DESIGN OF THE TRANSMISSION ELECTRON

MICROSCOPEMICROSCOPE Comparison of Light Microscope to Comparison of Light Microscope to

Transmission Electron MicroscopeTransmission Electron Microscope Alignment TheoryAlignment Theory Major Operational Modes of the Major Operational Modes of the

Transmission Electron MicroscopeTransmission Electron Microscope High ContrastHigh Contrast High ResolutionHigh Resolution DarkfieldDarkfield DiffractionDiffraction

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INTRODUCTIONINTRODUCTION A TRANSMISSION ELECTRON MICROSCOPE, or A TRANSMISSION ELECTRON MICROSCOPE, or TEM, has magnification and resolution capabilTEM, has magnification and resolution capabilities that are over ities that are over a thousanda thousand times beyond th times beyond that offered by the light microscope. at offered by the light microscope. It is an instrument that is used to reveal the It is an instrument that is used to reveal the ultultrastructurerastructure of plant and animal cells as well as of plant and animal cells as well as viruses and may provide an image of the very viruses and may provide an image of the very macromolecules that make up these biologicamacromolecules that make up these biological entities. l entities.

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INTRODUCTIONINTRODUCTION The TEM is a complex viewing system The TEM is a complex viewing system

equipped with equipped with a set of electromagnetic lensesa set of electromagnetic lenses used to control the used to control the imaging electronsimaging electrons in order in order to generate the to generate the extremely fine structural extremely fine structural detailsdetails that are usually recorded on that are usually recorded on photographic film. photographic film.

Since the illuminating electrons Since the illuminating electrons pass pass throughthrough the specimens, the information is said to be a the specimens, the information is said to be a transmittedtransmitted image. image.

The modern TEM can achieve magnifications The modern TEM can achieve magnifications of one million times with resolutions of of one million times with resolutions of 0.1 0.1 nmnm. .

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Basic Systems Making Up a Basic Systems Making Up a Transmission Electron Transmission Electron

MicroscopeMicroscope The transmission electron microscope (Figures The transmission electron microscope (Figures

6.20A, B and C) is made up of 6.20A, B and C) is made up of a number of a number of different systemsdifferent systems that are that are integrated to form one integrated to form one functional unitfunctional unit capable of orienting and imaging capable of orienting and imaging extremely thin specimens. extremely thin specimens.

The The illuminating systemilluminating system consists of the consists of the electron electron gun and condenser lensesgun and condenser lenses that give rise to and that give rise to and control the control the amount of radiation striking the amount of radiation striking the specimenspecimen. .

A A specimen manipulation systemspecimen manipulation system composed of composed of thethe specimen stage, specimen holders, and specimen stage, specimen holders, and related hardwarerelated hardware is necessary for orienting the is necessary for orienting the thin specimen outside and inside the microscope. thin specimen outside and inside the microscope.

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Basic Systems Making Up a Basic Systems Making Up a Transmission Electron Transmission Electron

MicroscopeMicroscope The The imaging systemimaging system includes the includes the objective,objective,

intermediate, and projectorintermediate, and projector lenses that are lenses that are involved in forming, focusing, and magnifying involved in forming, focusing, and magnifying the image on the viewing screen as well as the the image on the viewing screen as well as the cameracamera that is used to record the image. that is used to record the image.

A A vacuum systemvacuum system is necessary to remove is necessary to remove interfering air molecules from the column of interfering air molecules from the column of the electron microscope. In the descriptions the electron microscope. In the descriptions that follow, the systems will be considered that follow, the systems will be considered from the top of the microscope to the bottom. from the top of the microscope to the bottom.

See Tables 6.3 and 6.4. See Tables 6.3 and 6.4.

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TABLE 6.3 Major Column Components of the TEM*Component Synonyms Function of ComponentsIllumination System Electron Gun Gun, Source Generates electrons and provides first

coherent crossover of electron beam

Condenser Lens 1 C1, Spot Size Determines smallest illumination spot size on specimen (see Spot Size in Table 6.4)

Condenser Lens 2 C2, Brightness Varies amount of illumination on specimen—in combination with C1 (see Brightness in Table 6.4)

Condenser Aperture

C2 Aperture Reduces spherical aberration, helps control amount of illumination striking specimen

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Specimen Manipulation System

Specimen Exchanger Specimen Air Lock Chamber and mechanism for inserting specimen

holder

Specimen Stage Stage Mechanism for moving specimen inside column of microscope

Imaging System Objective Lens — Forms, magnifies, and focuses first image (see

Focus in Table 6.4)

Objective Aperture — Controls contrast and spherical aberration

Intermediate Lens Diffraction Lens Normally used to help magnify image from objective lens and to focus diffraction pattern

Intermediate Aperture Diffraction Aperture, Field Limiting Aperture

Selects area to be diffracted

Projector Lens 1 P1 Helps magnify image, possibly used in some diffraction work

Projector Lens 2 P2 Same as P1

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Observation and Camera Systems

Viewing

Chamber— Contains viewing screen for final image

Binocular Microscope

Focusing Scope Magnifies image on viewing screen for accurate focusing

Camera — Contains film for recording

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Illuminating SystemIlluminating SystemTABLE 6.4 Major Components on Control Panels of the TEM*

Component Synonyms Functions of Component

Filament Emission Effects emission of electrons upon heating

Bias — Adjusts voltage differential between filament and shield to regulate yield of electrons

High Voltage Reset HV, kV Reset Activates high voltage to gun

High Voltage Select HV, kV Select Selects amount of high voltage applied to gun

Magnification Control MAG Controls final magnification of image by activating combinations of imaging lenses

Brightness C2 Controls current to second condenser lens

Gun Tilt — Electronically tilts electron beam beneath gun

Gun Horizontal — Electronically translates electron beam beneath gun

Spot Size C1 Controls final illumination spot size on specimen

Objective Stigmator OBJ STIG Corrects astigmatism

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Focus Wobbler Focus Aid Helps focus accurately at low magnifications

Exposure Meter — Monitors illumination for accurate exposures

Vacuum Meter VAC Monitors vacuum levels in various parts of scope

Focusing Control Focus—fine, medium, coarse

Controls current to objective lens for accurate focusing of image

Brightness Center Illumination Centration Translates entire illumination system onto screen center

Condenser Stigmator COND STIG Corrects astigmatism in condenser lenses

Intermediate Stigmator

INT STIG Corrects astigmatism in intermediate lens

Bright/Dark — Selects brightfield or darkfield operating mode

Main — Main power switch to console

Main Evac EVAC Main switch to vacuum system

HV Wobbler HV Modulate Wobbles high voltage to locate voltage center for alignment

Objective Wobbler OBJ MODUL Wobbles current to objective lens for alignment

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Illuminating SystemIlluminating SystemThis system is situated at the top of This system is situated at the top of

the microscope column and consists the microscope column and consists of the of the electron gunelectron gun (composed of the (composed of the filament, shield, and anodefilament, shield, and anode) and the ) and the condenser lenses. condenser lenses.

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Illuminating SystemIlluminating SystemElectron Gun.Electron Gun. Within the electron gun (Figure 6.21), the Within the electron gun (Figure 6.21), the filamentfilament

serves as the source of electrons. serves as the source of electrons. The standard filament, or The standard filament, or cathodecathode (Figure 6.22), is (Figure 6.22), is

composed of a composed of a V-shaped tungsten wire V-shaped tungsten wire approximately 0.1 mm in diameterapproximately 0.1 mm in diameter (about the (about the thickness of a human hair). thickness of a human hair).

Being a metal, tungsten contains Being a metal, tungsten contains positive ions and positive ions and free electronsfree electrons that are strongly attracted to the that are strongly attracted to the positive ions. positive ions.

Fortunately, it is possible to Fortunately, it is possible to entice the outermost entice the outermost orbitalorbital, , or valenceor valence, electrons out of the tungsten by , electrons out of the tungsten by first applying a high voltage to the filament and then first applying a high voltage to the filament and then heating the metal by running a small amount of DC heating the metal by running a small amount of DC electrical current through the filament while electrical current through the filament while operating within a vacuum. operating within a vacuum.

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Page 169Page 169

Figure 6.21(A) Diagram of an electron gun showingfilament, shield, and anode. The shield is connected directly to the high voltage, whereas the high voltage leading to the filament has a variable resistor (VR) to vary the amount of high voltage. The output from the variable resistor is then passed through two balancing resistors (BR) which are attached to the filament. (B) Actual electron gun from TEM showing filament (f), shield (s), and anode (a). Compare to line drawing in 6.21(A).

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Figure 6.22Standard V-shaped tungsten filament (f) used in mostelectron microscopes. The filament is spotwelded tothe larger supporting arms, which pass through theceramic (c) insulator and plug into the electrical leads ofthe gun.

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Illuminating SystemIlluminating System In other words, a certain amount of In other words, a certain amount of energyenergy

must be put into the system to cause the must be put into the system to cause the electrons to leave the filamentelectrons to leave the filament. .

The amount of The amount of energy necessaryenergy necessary to bring to bring about electron emission is termed the about electron emission is termed the work functionwork function of the metal. of the metal.

Although tungsten has a relatively high Although tungsten has a relatively high work function, it has an work function, it has an excellent yield of excellent yield of electronselectrons just just below its rather high melting below its rather high melting pointpoint of of 3,653° K. 3,653° K.

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Illuminating SystemIlluminating System In practical terms, one first applies a fixed amount of In practical terms, one first applies a fixed amount of

negative high voltagenegative high voltage (typically 50, 75 or 100 kV) and (typically 50, 75 or 100 kV) and then slowly then slowly increases the amount of direct currentincreases the amount of direct current running through the filament to running through the filament to heat it to achieve the heat it to achieve the emission of electronsemission of electrons ( (thermionic emissionthermionic emission).).

As one applies more heat to the filament, the yield of As one applies more heat to the filament, the yield of electrons increases until electrons increases until the filament begins to melt the filament begins to melt and evaporate in the high vacuum of the microscopeand evaporate in the high vacuum of the microscope (Figure 6.23). (Figure 6.23).

At some optimal temperature, the gun achieves good At some optimal temperature, the gun achieves good electron emission as well as an acceptable filament electron emission as well as an acceptable filament life: this is termed the life: this is termed the saturation pointsaturation point (discussed (discussed later). later).

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Figure 6.23The importance of proper saturation of the filament isshown in these two curves. (a) Solid line shows therelationship between electron emission from a filament asa function of the temperature of the filament. Theoptimal temperature is 2,600° K. (b) When one exceeds2,600° K filament temperature, the filament life dropsdramatically (dashed line).

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Illuminating SystemIlluminating System By examining Figure 6.23, it becomes apparent that a good electron yielBy examining Figure 6.23, it becomes apparent that a good electron yield is achieved at d is achieved at 2,600° K2,600° K where the filament life is around where the filament life is around 100 hours. 100 hours. OversaturatingOversaturating the gun by heating the filament even the gun by heating the filament even 200° K200° K beyond 2,60 beyond 2,600° K results in a dramatic decrease in filament life. 0° K results in a dramatic decrease in filament life. The average life of a filament ranges from 25 hours in older electron micThe average life of a filament ranges from 25 hours in older electron microscopes to over 200 hours in microscopes with good vacuum systems aroscopes to over 200 hours in microscopes with good vacuum systems and scrupulously maintained gun areas. nd scrupulously maintained gun areas. The The major causes of premature filament failuremajor causes of premature filament failure are: are:

oversaturation, oversaturation, high voltage discharge caused by dirt in the gun region,high voltage discharge caused by dirt in the gun region, poor vacuum, and poor vacuum, and air leaks or outgassing from contaminants in the gun chambeair leaks or outgassing from contaminants in the gun chamber. r.

The two controls on the panel of the microscope that are used to initiate The two controls on the panel of the microscope that are used to initiate the flow of electrons are usually labeled the flow of electrons are usually labeled accelerating voltageaccelerating voltage (or possibl (or possibly kV, HV or HT) and y kV, HV or HT) and emissionemission or or saturationsaturation (or sometimes (or sometimes filament).filament).

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Illuminating SystemIlluminating System If one induces the If one induces the emission of electronsemission of electrons from a filame from a filament as described above, this will result in the emanationt as described above, this will result in the emanation of electrons n of electrons in all directionsin all directions. . Without a mechanism for Without a mechanism for guiding themguiding them, most of the el, most of the electrons would not enter the ectrons would not enter the illuminating systemilluminating system. . A A secondsecond part of the electron gun, the part of the electron gun, the shieldshield (also call(also called Wehnelt cylinder, bias shield, or grid cap), is involved Wehnelt cylinder, bias shield, or grid cap), is involved in assuring that the majority of the electrons go in ted in assuring that the majority of the electrons go in the proper direction. he proper direction. The shield is a caplike structure that covers the filameThe shield is a caplike structure that covers the filament and is maintained at a nt and is maintained at a slightly more negative voltagslightly more negative voltagee potential than the filament. potential than the filament.

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Illuminating SystemIlluminating System Because it is several Because it is several hundred volts more hundred volts more negativenegative tha than the n the 50 to 100 kV electrons50 to 100 kV electrons, the shield surrounds the e, the shield surrounds the electrons with a lectrons with a repulsive fieldrepulsive field that is breachable only t that is breachable only through hrough a 2 to 3 mm aperturea 2 to 3 mm aperture directly in front of the fila directly in front of the filament tip. ment tip. Electrons exit the shield aperture and are drawn towarElectrons exit the shield aperture and are drawn toward an apertured disc, or anode, the third part of the elecd an apertured disc, or anode, the third part of the electron gun (Figure 6.21). tron gun (Figure 6.21). The anode is connected to ground so that the highly nThe anode is connected to ground so that the highly negative electrons are strongly attracted to it. egative electrons are strongly attracted to it. Thus it is Thus it is positive with respect to the gunpositive with respect to the gun. . In fact, the In fact, the highly attractive pullhighly attractive pull of the of the anode anode in combiin combination with the nation with the negative surfacenegative surface of the shield act as an of the shield act as an electrostatic "lens" to electrostatic "lens" to generate a crossover imagegenerate a crossover image of th of the electron source near the anode (Figure 6.24). e electron source near the anode (Figure 6.24).

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Figure 6.21(A) Diagram of an electron gun Showing filament, shield, and anode. The shield is connected directly to the high voltage, whereas the high voltage leading to thefilament has a variable resistor (VR) to vary the amount of high voltage. The output from the variable resistor is then passed through two balancing resistors (BR) Which are attached to the filament. (B) Actual electron gun from TEM showing filament (f), shield (s), and anode (a). Compare to line drawing in 6.21(A).

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Figure 6.24 The self-biased electron gun. The shield (Wehnelt cylinder) is slightly more negative than the filament to control the release of electrons from the gun.

A variable bias resistor (see Figure 6.21A) regulates the degree of negativity of the filament.

The anode serves as a positive attracting force and serves as an electrostatic lens (in combination with the shield) to help focus the electrons into a crossover spot approximately 50 μm across.

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Illuminating SystemIlluminating System Just as electromagnetic lenses have Just as electromagnetic lenses have

two two forces or polesforces or poles, , electrostaticelectrostatic lenses have lenses have positively and negativelypositively and negatively charged surfaces to attract or repel charged surfaces to attract or repel and, thereby, focus electrons. and, thereby, focus electrons.

The term The term crossovercrossover refers to the point refers to the point where the where the electrons focus or convergeelectrons focus or converge and cross over and cross over each other's pathseach other's paths. .

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Illuminating SystemIlluminating System Variable Self-Biased Gun.Variable Self-Biased Gun. It was stated It was stated

earlier that the earlier that the high voltage shieldhigh voltage shield is is slightly more negativeslightly more negative than the than the filament. filament.

This difference in negative potential, or This difference in negative potential, or biasbias,, is established by connecting the is established by connecting the shield directly to the negative high voltage shield directly to the negative high voltage line while placing a variable resistor in the line while placing a variable resistor in the high voltage line to the filament (Figure high voltage line to the filament (Figure 6.21A). 6.21A).

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Illuminating SystemIlluminating System By By varying the value of this resistorvarying the value of this resistor, the , the

filament may be made less negative than filament may be made less negative than the shield (usually by the shield (usually by 100 to 200 volts100 to 200 volts). The ). The greater the value of the variable bias greater the value of the variable bias resistor, the less negative the filament will resistor, the less negative the filament will become. become.

As the filament becomes As the filament becomes less negativeless negative, , fewer electrons will be able to pass throughfewer electrons will be able to pass through the shield aperture since they are now the shield aperture since they are now repulsed to a greater degree by the shield. repulsed to a greater degree by the shield.

The overall effect of the The overall effect of the variable bias,variable bias, therefore, is to regulate the escape of therefore, is to regulate the escape of electrons through the shield aperture. electrons through the shield aperture.

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Illuminating SystemIlluminating System In addition to In addition to high voltagehigh voltage, one applies a certain , one applies a certain

amount of amount of direct current to the filamentdirect current to the filament in order to in order to heat upheat up the filament and enhance electron emission. the filament and enhance electron emission.

As this As this current passescurrent passes through the variable bias through the variable bias resistorresistor, a certain amount of , a certain amount of voltage voltage is generated and is generated and applied to the applied to the shieldshield in order to make it more in order to make it more negativenegative. .

Therefore, as one continues to Therefore, as one continues to increase the heating increase the heating current to the filamentcurrent to the filament, the , the numbers of electrons numbers of electrons coming off the filament will increasecoming off the filament will increase. .

But since the But since the shield shield is becoming progressively is becoming progressively negativenegative, the , the total number of electronstotal number of electrons actually actually passing through the shield aperturepassing through the shield aperture does not increase does not increase significantly. significantly.

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Illuminating SystemIlluminating System The so-called The so-called saturation pointsaturation point of the gun is the point wher of the gun is the point where the e the number of electrons emitted from the gunnumber of electrons emitted from the gun no no longer ilonger increasesncreases as the filament is heated. as the filament is heated. The gun is, therefore, said to be The gun is, therefore, said to be self-biasingself-biasing, since it throttl, since it throttles back es back on electron emissionon electron emission as the heat is increased. as the heat is increased. It is important that the operator realize that It is important that the operator realize that increasing the increasing the heat of the filament beyond the saturation pointheat of the filament beyond the saturation point will will not not inincrease the crease the brightness of the gunbrightness of the gun but will considerably but will considerably shortshorten the filament lifeen the filament life.. On the other hand, On the other hand, undersaturationundersaturation of the filament may lea of the filament may lead to d to instabilities in the illuminationinstabilities in the illumination of the specimen and ca of the specimen and cause problems if analytical procedures (such as X-ray analysiuse problems if analytical procedures (such as X-ray analysis) are to be attempted. s) are to be attempted. The arrangement for The arrangement for controlling electron emissioncontrolling electron emission in mode in modern electron microscopes is termed the rn electron microscopes is termed the variable self-biased variable self-biased gungun..

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Illuminating SystemIlluminating System Controlling the Amount of Illumination Striking Controlling the Amount of Illumination Striking

the Specimen.the Specimen. It is possible to make practical use It is possible to make practical use of the variable bias to regulate the amount of of the variable bias to regulate the amount of illumination that strikes the specimen. illumination that strikes the specimen.

For example, when operating at For example, when operating at high magnificationshigh magnifications with with small condenser spot sizessmall condenser spot sizes, it may be necessary , it may be necessary to to alter the biasalter the bias to effect greater to effect greater gun emissionsgun emissions. .

Of course, the filament life will be shortened, but Of course, the filament life will be shortened, but this may this may be necessarybe necessary in order to in order to critically view and critically view and focus the specimenfocus the specimen. .

It is also important to remember that the It is also important to remember that the greater the greater the beam currentbeam current, the greater the specimen , the greater the specimen damagedamage. .

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Illuminating SystemIlluminating System Moving the Moving the filament closer to the shield aperturefilament closer to the shield aperture

will will permit more electronspermit more electrons to pass through to the to pass through to the condenser lensescondenser lenses. .

However, if the filament is placed too close to the However, if the filament is placed too close to the aperture, the aperture, the bias control by the shield will be bias control by the shield will be lostlost, and the emission will become , and the emission will become excessiveexcessive. . Filaments placed too Filaments placed too far far away from the shield away from the shield aperture, on the other hand, may aperture, on the other hand, may never yield never yield sufficient numberssufficient numbers of electrons from the gun. of electrons from the gun.

Therefore, careful placement of the filament Therefore, careful placement of the filament relative to the shield aperture is very important relative to the shield aperture is very important and should be in accordance with the and should be in accordance with the manufacturer's specifications manufacturer's specifications

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Figure 6.24 The self biased electron gun. The shield (Wehnelt cylinder) is slightly more negative than thefilament to control the release of electrons from the gun.A variable bias resistor (see Figure 6.21A) regulates the degree of negativity of the filament. The anode serves as a positive attracting force and serves as an electrostatic lens (in combination with the shield) to help focus the electrons into acrossover spot approximately 50μm across.

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Illuminating SystemIlluminating System The The distance distance of theof the anode anode from the filament and from the filament and

shield is also important. shield is also important. As one moves the As one moves the anode closeranode closer to the filament, to the filament,

more electronsmore electrons will be will be extracted from the gunextracted from the gun. . This becomes a consideration when using This becomes a consideration when using lower lower

accelerating voltagesaccelerating voltages where it may be necessary to where it may be necessary to move the anode closer to assist in the extraction of move the anode closer to assist in the extraction of the lower energy electrons (50 kV, for example). the lower energy electrons (50 kV, for example).

Some electron microscopes have an Some electron microscopes have an external external adjustment screwadjustment screw that will mechanically adjust the that will mechanically adjust the height of the anode, while other models have a height of the anode, while other models have a pneumatically actuated "anode lifter" that changes pneumatically actuated "anode lifter" that changes in response to the kilovolt selected by the operator. in response to the kilovolt selected by the operator.

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Illuminating SystemIlluminating System The choice of The choice of kV kV should be considered carefullshould be considered carefully. y. Lower kVs such as 50 kV will generate an imagLower kVs such as 50 kV will generate an image with e with higher contrast but lower resolutionhigher contrast but lower resolution, w, while higher hile higher kVs (100–125 kV)kVs (100–125 kV) improve improve resolutioresolutionn but but lower overall contrastlower overall contrast. . Less specimen damageLess specimen damage will result at the will result at the higher higher kVs since the kVs since the speedier electronsspeedier electrons interact for interact for a shorter period of time with the specimena shorter period of time with the specimen..

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Illuminating SystemIlluminating System Other Gun Designs.Other Gun Designs. The filament shape may be The filament shape may be

altered as illustrated in Figure 6.25b, where the tip altered as illustrated in Figure 6.25b, where the tip was first flattened and then sharpened to a point. was first flattened and then sharpened to a point.

It is also possible to purchase a pointed filament It is also possible to purchase a pointed filament made by welding a single crystal of tungsten onto made by welding a single crystal of tungsten onto the curved tip of a standard filament (Figure 6.25c). the curved tip of a standard filament (Figure 6.25c).

Both types of Both types of pointed filamentspointed filaments have a considerably have a considerably shorter lifetimeshorter lifetime than do standard filaments. than do standard filaments.

However, since the However, since the initial gun crossover imageinitial gun crossover image is is much much smallersmaller and the and the beam is highly coherentbeam is highly coherent, they , they are necessary for are necessary for high resolution studieshigh resolution studies where where beam damage may be a consideration (e.g., viewing beam damage may be a consideration (e.g., viewing crystalline lattice planes). crystalline lattice planes).

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Figure 6.25Drawing of three different filament tips. (a) Standard V-shaped filament tip. (b) Standard filament tip that was flattened and then sharpened to a fine point. (c) Filament tip where a crystal of tungsten was spotwelded onto the curved end.

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Illuminating System: Illuminating System: LaBLaB66

Besides being made of tungsten, filaments maBesides being made of tungsten, filaments may also be constructed of y also be constructed of lanthanum hexaboridlanthanum hexaboridee,, which has which has a lower work functiona lower work function. . Typically, these filaments operate at temperatTypically, these filaments operate at temperatures ures 1,000° K lower than tungsten and1,000° K lower than tungsten and have a have a bbrightness several times greaterrightness several times greater than a standar than a standard tungsten source. d tungsten source. The lifetime of such filaments ranges from The lifetime of such filaments ranges from 700 700 to 2,000 hoursto 2,000 hours. This type of filament may be m. This type of filament may be made from a ade from a single LaBsingle LaB66 crystal crystal with one end ha with one end having a ving a point measuring only several micrometpoint measuring only several micrometers acrossers across (Figure 6.26). (Figure 6.26).

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Figure 6.26Lanthanum hexaboride cathode. The crystal (C) is held in place by means of pyrolytic graphite (G) blocks with compressive force generated by molybdenum (M) alloy posts designed to withstand extremely high temperatures.

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Illuminating SystemIlluminating System LaBLaB66 filaments are coming into use slowly, since they a filaments are coming into use slowly, since they are considerably re considerably more expensive than tungsten filamemore expensive than tungsten filamentsnts and are extremely chemically reactive when hot. F and are extremely chemically reactive when hot. For the latter reason, vacuums greater than or the latter reason, vacuums greater than 1010-5-5 Pa are Pa are essentialessential (see section "Vacuum System"), and special (see section "Vacuum System"), and special filament mounts must be constructed from such nonrfilament mounts must be constructed from such nonreactive elements as rhenium or vitreous carbon. eactive elements as rhenium or vitreous carbon. LaBLaB66 filaments are useful when filaments are useful when small beam crossover small beam crossover sizessizes containing large containing large numbers of electrons are necesnumbers of electrons are necessarysary— as in high magnification/resolution studies, for — as in high magnification/resolution studies, for elemental analysis, or in high resolution scanning elecelemental analysis, or in high resolution scanning electron microscopy. tron microscopy.

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Illuminating System: Illuminating System: cold field emission guncold field emission gun

A totally different gun, nearly a A totally different gun, nearly a thousand times thousand times brighter than the standard gunbrighter than the standard gun, may also be used , may also be used under under certain conditionscertain conditions. .

In the In the cold field emission guncold field emission gun,, the filament is a the filament is a single crystal of tungstensingle crystal of tungsten with its with its atomic atomic crystalline lattice precisely orientedcrystalline lattice precisely oriented to maximize to maximize electron emission. electron emission.

Electrons are Electrons are notnot generated by generated by thermionic thermionic emission (heatingemission (heating), but are actually drawn out of ), but are actually drawn out of the tungsten crystal by the tungsten crystal by a series of positive high a series of positive high voltage anodesvoltage anodes that act as that act as electrostatic lenseselectrostatic lenses to to focus the gun focus the gun crossover to a spot size of 10 nmcrossover to a spot size of 10 nm (Figure 6.27). (Figure 6.27).

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Figure 6.27The field emission gun. Electrons are extracted from a single crystal of tungsten by a series of anodes that are made several thousand volts positive. It is not necessary to heat this type of filament.

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Illuminating System: Illuminating System: cold field emission guncold field emission gun

A major disadvantage of the A major disadvantage of the cold field cold field emission gun is the ultrahigh vacuum required emission gun is the ultrahigh vacuum required (greater than 10(greater than 10-8 -8 Pa)Pa) and the extreme and the extreme susceptibility of the filament to contaminants. susceptibility of the filament to contaminants.

Cold field emission guns are very useful in Cold field emission guns are very useful in high resolution scanninghigh resolution scanning and and scanning scanning transmissiontransmission electron microscopes and are electron microscopes and are now being incorporated into now being incorporated into high resolution high resolution transmission electron microscopes.transmission electron microscopes. See Table See Table 6.56.5 for a comparison of the three major for a comparison of the three major filaments. filaments.

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Page 172TABLE 6.5 Comparison of the Three Major Filaments in Terms of Brightness, Size of the Source Crossover, Energy Spread, Service Life, and Vacuum Required Cold Field Emission Lanthanum Hexab

orideTungsten Filament

Brightness (A/cm2 __ sr) 109 107 106

Source Diameter (nm) <10 104 >104

Energy Spread (eV) 0.2–0.3 1.0–2.0 1.0–2.0

Service Life (hours) >2,000 1,000–2,000 40–100

Vacuum Required (Pa) 10-8 10-5 10-3

((Courtesy D. Rathkey, FEI Company.)Courtesy D. Rathkey, FEI Company.)

4949

Illuminating System- Illuminating System- Condenser Condenser LensesLenses

Condenser LensesCondenser Lenses.. This second major part of the illu This second major part of the illuminating system gathers the electrons of the first crosminating system gathers the electrons of the first crossover image from the gun and sover image from the gun and focuses electrons onto tfocuses electrons onto the specimenhe specimen.. Modern transmission electron microscopes have Modern transmission electron microscopes have two two condenser lensescondenser lenses, unlike the first microscopes that ha, unlike the first microscopes that had only one. The d only one. The first condenser lens (designated C1first condenser lens (designated C1) is ) is a a demagnifying lensdemagnifying lens that that decreases the size of the 50 decreases the size of the 50 μμmm gun gun crossovercrossover to to generate a range of spot sizesgenerate a range of spot sizes fro from m 20 20 μμm down to 1 m down to 1 μμmm. . The The second condenser lens (C2),second condenser lens (C2), on the other hand, on the other hand, enenlarges the C1 spotlarges the C1 spot. The . The overall effect of both lensesoverall effect of both lenses is t is to o control precisely the amount of electron irradiationcontrol precisely the amount of electron irradiation or illumination or illumination striking the specimenstriking the specimen. .

5050

Illuminating System-Illuminating System- Condenser Condenser LensesLenses

The operating principle for using C1 and C2 is to The operating principle for using C1 and C2 is to ggenerate a spot on the specimenenerate a spot on the specimen of the proper size of the proper size to to illuminate only the area being examinedilluminate only the area being examined.. Therefore, at higher magnifications Therefore, at higher magnifications smaller spot ssmaller spot sizes should be focused on the specimenizes should be focused on the specimen (Figure 6. (Figure 6.28B), while 28B), while larger spotslarger spots may be used at lower ma may be used at lower magnifications (Figure 6.28A). gnifications (Figure 6.28A). Because spot sizes are controlled, beam damage Because spot sizes are controlled, beam damage can be minimized to parts of the specimen not becan be minimized to parts of the specimen not being viewed. ing viewed. This offered a great advantage when TEMs were This offered a great advantage when TEMs were made with two condenser lenses rather than one. made with two condenser lenses rather than one.

5151

Figure 6.28The condenser lens system. (A) In this mode, the 50 μμm gun crossover is reduced to 5 μμm by condenser lens 1, C1, and then slightly enlarged by condenser lens 2, C2, to yield a 10 μμm spot on the specimen that is five times brighter than the initial gun crossover.

5252

Figure 6.28 (B) At higher magnifications, the 50 μμm gun crossover is reduced to 1.5 μμm by a highly energized C1. This refracts the peripheral electrons to such a great angle that they cannot enter C2 and are therefore lost.

After C2 slightly enlarges the C1 spot, the resulting 2 μμm spot is rather dim.

5353


Suppose one is working at a magnification of 50,Suppose one is working at a magnification of 50,000X. 000X. At this high magnification, the C1 lens should be At this high magnification, the C1 lens should be highly energized to highly energized to demagnify the 50 demagnify the 50 μμm illumim illumination spotnation spot from the gun down to from the gun down to 1 to 2 1 to 2 μμmm. . Next, the C2 lens should be used to adjust the siNext, the C2 lens should be used to adjust the size of the ze of the C1 illumination spot to cover only the sC1 illumination spot to cover only the specimen area being viewedpecimen area being viewed. Since the average v. Since the average viewing screen is about iewing screen is about 100 mm across100 mm across, a , a 2 2 μμmm s spot of illumination enlarged 50,000X would just pot of illumination enlarged 50,000X would just cover the screen (2 μm X 50,000 = 100 mm). cover the screen (2 μm X 50,000 = 100 mm).

5454


Therefore, the C2 lens should also be highly enTherefore, the C2 lens should also be highly energized to generate a 2 μm spot on the specimergized to generate a 2 μm spot on the specimen. en. At a magnification of 10,000X, it is possible to kAt a magnification of 10,000X, it is possible to keep C1 highly energized but to use eep C1 highly energized but to use C2 toC2 to magni magnify or fy or spread outspread out the 2 μm spot an additional 5X the 2 μm spot an additional 5X to just cover the 100 mm screen. to just cover the 100 mm screen. However, the illumination will be about 5 timeHowever, the illumination will be about 5 times dimmer. It is important, however, to keep ths dimmer. It is important, however, to keep the C2 lens spread out away from crossover to me C2 lens spread out away from crossover to minimize specimen damage but with enough illuinimize specimen damage but with enough illumination to focus. mination to focus.

5555

Illuminating System-Illuminating System- Condenser Condenser LensesLenses If one studies Figure 6.28, it is If one studies Figure 6.28, it is

apparent why smaller spot sizes apparent why smaller spot sizes are necessarily dimmer. are necessarily dimmer.

If C1 is highly energized in order If C1 is highly energized in order to generate a small spot (Figure to generate a small spot (Figure 6.28B), the focal length is made 6.28B), the focal length is made so shortso short and the aperture angle and the aperture angle so greatso great that many electrons are that many electrons are refracted to such an extent that refracted to such an extent that they do not enter C2. they do not enter C2.

On the other hand, if On the other hand, if C1 is C1 is weakened to generateweakened to generate a a larger larger spotspot, the focal length is longer , the focal length is longer and the aperture angle is and the aperture angle is smaller so that effectively all smaller so that effectively all electrons may now enter C2 electrons may now enter C2 (Figure 6.28A). (Figure 6.28A).

Therefore, as the Therefore, as the C1 spot is C1 spot is made progressively smallermade progressively smaller, , overall illumination tends to overall illumination tends to diminish. diminish.

5656


This poses a problem at higher magnifications where This poses a problem at higher magnifications where very small spot sizes are neededvery small spot sizes are needed. . Illumination may become so Illumination may become so dimdim that microscopists that microscopists must allow must allow 30 to 60 minutes30 to 60 minutes for their eyes to adapt to for their eyes to adapt to working under such dark conditions. working under such dark conditions. However, it may be possible to However, it may be possible to increase the illuminatiincrease the illumination on the specimenon on the specimen using the techniques described pr using the techniques described previously in the chapter section entitled 'eviously in the chapter section entitled ''Controlling t'Controlling the Amount of Illumination Striking the Specimenhe Amount of Illumination Striking the Specimen," or ," or by by using high sensitivity electronic cameras to view diusing high sensitivity electronic cameras to view dimly illuminated specimensmly illuminated specimens. .

5757


Apertures in Condenser LensesApertures in Condenser Lenses.. Depending o Depending on the design of the transmission electron micrn the design of the transmission electron microscope, one or both condenser lenses may haoscope, one or both condenser lenses may have ve apertures of variable sizesapertures of variable sizes. . Generally, the C1 aperture is Generally, the C1 aperture is an internal apertan internal aperture of a fixed sizeure of a fixed size, while the , while the C2 C2 aperture is aperture is varivariableable by inserting into the by inserting into the electron beam pathelectron beam pathway aperturesway apertures of different sizes attached to th of different sizes attached to the end of a shaft. e end of a shaft. A popular method is to use a molybdenum foil A popular method is to use a molybdenum foil strip containing 3 or 4 holes of 500, 300, 200, astrip containing 3 or 4 holes of 500, 300, 200, and 100 nd 100 μμm in diameter (Figure 6.29). m in diameter (Figure 6.29).

5858

Figure 6.29Variable aperture holder from a TEM. The rod contains a molybdenum strip (m) with apertures of various sizes.Positioning screws (s) permit the precise alignment of the apertures in the electron beam. An O-ring seal (o) permits the aperture to be sealed off inside the vacuum of the microscope column. Insert shows enlargement of the molybdenum aperture strip held in place by a brass retainer clip. Arrows point to apertures in the strip.

5959


Larger condenser apertures permitLarger condenser apertures permit most of the most of the electrons to pass through the lenselectrons to pass through the lens and, therefore, and, therefore, yield a brighter spot on the specimenyield a brighter spot on the specimen. .

Smaller apertures cut out more peripheral electronsSmaller apertures cut out more peripheral electrons and, hence, reduce the and, hence, reduce the illumination on the specimenillumination on the specimen. .

However, since spherical aberration is concomitantly However, since spherical aberration is concomitantly reduced, reduced, greater resolution is possible using smaller greater resolution is possible using smaller condenser aperturescondenser apertures. .

The operational principle to remember is The operational principle to remember is larger larger condenser apertures give more illumination but with condenser apertures give more illumination but with more spherical aberration.more spherical aberration.

6060

Specimen Manipulation Specimen Manipulation SystemSystem

Most biological specimens are mounted on a Most biological specimens are mounted on a copper meshwork or copper meshwork or gridgrid..

Grids are placed into a Grids are placed into a specimen holderspecimen holder and, and, after after insertion into an air lockinsertion into an air lock, the chamber , the chamber is evacuated and the specimen holder is is evacuated and the specimen holder is inserted into the stage of the microscopeinserted into the stage of the microscope. .

(In very old microscopes, no air locks were (In very old microscopes, no air locks were provided, so it was necessary to admit air to provided, so it was necessary to admit air to the entire column in order to insert a the entire column in order to insert a specimen. Such changes would take 5 to 10 specimen. Such changes would take 5 to 10 minutes versus 30 or so seconds in modern minutes versus 30 or so seconds in modern air-locked microscopes.) air-locked microscopes.)

6161


The The specimen stagespecimen stage is a micromanipulator for moving is a micromanipulator for moving the specimen in the specimen in x and y directionsx and y directions in increments as sm in increments as small as all as 10 nm10 nm, the width of a cell membrane. , the width of a cell membrane. Depending on the design of the specimen holder and sDepending on the design of the specimen holder and stage, it may also be possible to tage, it may also be possible to tilt and rotate the spectilt and rotate the specimen inside the column of the electron microscopeimen inside the column of the electron microscope. . Some of the newer micro-processor-controlled TEMs Some of the newer micro-processor-controlled TEMs have have automated stage controlsautomated stage controls that that permit motorized permit motorized and precise movement of the specimenand precise movement of the specimen. . An important feature of such computer-controlled staAn important feature of such computer-controlled stages is the ability to ges is the ability to memorize specified coordinatesmemorize specified coordinates an and to be able to return to these locations on command. d to be able to return to these locations on command.

6262


Top-Entry StageTop-Entry Stage.. One type of One type of specimen specimen holderholder is a is a brass cartridgebrass cartridge with a with a long long cylinder that enters the objective lenscylinder that enters the objective lens as as the holder is placed in the the holder is placed in the stage on top stage on top of the objective lensof the objective lens (Figure 6.30). (Figure 6.30).

The The grid sitsgrid sits on the on the end of the cylinderend of the cylinder and is and is held firmlyheld firmly in place by a tight- in place by a tight-fitting sleeve that slips over the cylinder. fitting sleeve that slips over the cylinder.

6363

Figure 6.30(left) Short, top-entry grid holder for high contrast,low-magnification work. Resolution is not as good withthis type of grid holder since the specimen is placedhigher in the objective lens, necessitating a longer focallength of the lens. (right) Standard top-entry specimengrid holder for high resolution work. The specimen gridis placed on the end of the grid holder shaft and held inplace with a sleeve that is slipped over the shaft (arrow).These holders are placed in the specimen stage with thegrids in the downward position in the polepiece.

6464


Firm contact between Firm contact between grid and specimen holdergrid and specimen holder is is essential in order to essential in order to dissipate the buildup of heat dissipate the buildup of heat and static charges resulting from bombardment and static charges resulting from bombardment by the electron beamby the electron beam. .

To optimize this dissipation, insert the To optimize this dissipation, insert the copper copper mesh facing the electron beammesh facing the electron beam (i.e., specimen (i.e., specimen down). down).

However, be careful that the However, be careful that the specimen and specimen and supporting membranesupporting membrane are well are well adhered to the adhered to the copper gridcopper grid, or they may detach and fall onto the , or they may detach and fall onto the microscope stage or objective lens, necessitating microscope stage or objective lens, necessitating time-consuming disassembly of the microscope. time-consuming disassembly of the microscope.

6565


After the After the grid has been inserted in the specimen grid has been inserted in the specimen holderholder, the , the cartridge is placed in an air lockcartridge is placed in an air lock where where the air is removed by the vacuum system. the air is removed by the vacuum system.

The air lock is opened to the high vacuum of the The air lock is opened to the high vacuum of the microscope and the microscope and the specimen holder transferredspecimen holder transferred onto the onto the movable stagemovable stage of the microscope. of the microscope.

It is again important that the contact between the It is again important that the contact between the specimen holder and brass stage be firmspecimen holder and brass stage be firm in order in order to dissipate any static charges. to dissipate any static charges.

For instance, a dirty specimen stage or holder will For instance, a dirty specimen stage or holder will prevent such contact and may lead to thermal or prevent such contact and may lead to thermal or electrostatic drift in the specimen. electrostatic drift in the specimen.

Dirty specimen holders are a major cause of Dirty specimen holders are a major cause of specimen drift in the TEMspecimen drift in the TEM. . Holders must be Holders must be examined and cleaned on a regular basis.examined and cleaned on a regular basis.

6666


Some top-entry holders are designed to Some top-entry holders are designed to tilt tilt mechanically several degreesmechanically several degrees, and some stages have , and some stages have gearing that will enable the specimen to be gearing that will enable the specimen to be rotated rotated 360 degrees360 degrees. .

It is possible to vary the It is possible to vary the length of the cartridgelength of the cartridge nosepiece and construct holdersnosepiece and construct holders suited to special suited to special purposes. purposes.

For instance, short For instance, short nosepiece holders are usefulnosepiece holders are useful for for high high contrast and low magnificationcontrast and low magnification applications applications ((longer focal length objective lenslonger focal length objective lens), while ), while longer longer nosepieces are suitable for high resolution studiesnosepieces are suitable for high resolution studies (short focal length objective lens). (short focal length objective lens).

Although it is possible to insert only one cartridge Although it is possible to insert only one cartridge holder into the stage, some microscopes have air holder into the stage, some microscopes have air locks that accommodate several cartridges so that it locks that accommodate several cartridges so that it is not necessary to break vacuum in order to insert is not necessary to break vacuum in order to insert another specimen (Figure 6.31). another specimen (Figure 6.31).

6767

Figure 6.31Multiple grid holder specimen air lock. In this design, itis possible to load six grid holders into the air lock,evacuate the sealed chamber, select the holder, andinsert it into the column using the exchanger arm shown.

6868


Side-Entry StageSide-Entry Stage.. In this type of stage, the In this type of stage, the spspecimen grid is introduced into the microscope ecimen grid is introduced into the microscope stagestage by entering through the by entering through the side of the objecside of the objective lens polepiecetive lens polepiece. . The specimen holder resembles an The specimen holder resembles an aperture haperture holder consisting of a rodolder consisting of a rod with a with a flat plate on onflat plate on one ende end that has one or more recessed areas for that has one or more recessed areas for holding grids (Figure 6.32).holding grids (Figure 6.32).

6969

Figure 6.32(A) Side-entry, multiple specimen grid holder.

K = specimen selection knob for positioning proper grid, s = specimen holder area where grids are inserted and held.

(B) Close-up view of side-entry, multiple grid holder showing a specimen grid in the countersunk depression on the right (arrow). The grids are held in place by meansof the plate shown in the prongs of the forceps. The plate is positioned over the grids and held in place by a spring-loaded latch.

7070


The grid is placed into the The grid is placed into the flanged recessflanged recess and held in p and held in position with a osition with a clip or plateclip or plate (Figure 6.32B). (Figure 6.32B). The specimen rod may then be The specimen rod may then be evacuated in the air loevacuated in the air lockck and the and the rod inserted into the microscope stagerod inserted into the microscope stage. . With some types of rods, the end bearing the specimeWith some types of rods, the end bearing the specimen grids may be inserted into the stage and detached wn grids may be inserted into the stage and detached while the carrier rod remains in the air lock. hile the carrier rod remains in the air lock. Most types of rods are inserted into the specimen stagMost types of rods are inserted into the specimen stage and remain in the stage during the viewing process. e and remain in the stage during the viewing process. Since the rod enters through the side of the polepiece, Since the rod enters through the side of the polepiece, it is necessary to design the polepiece with a large enoit is necessary to design the polepiece with a large enough gap to permit entry and allow for tilting of the speugh gap to permit entry and allow for tilting of the specimen by as much as 65 degrees from the horizontal.cimen by as much as 65 degrees from the horizontal.

7171


Side-entry stages provide much Side-entry stages provide much more versatile manipulation more versatile manipulation of the specimenof the specimen. . Besides the standard x and y horizontal movements, the specBesides the standard x and y horizontal movements, the specimen holder may permit imen holder may permit tilting, rotation, a second axis of tilttilting, rotation, a second axis of tilt (double-tilt stage), and special modifications as described in (double-tilt stage), and special modifications as described in the next paragraph. the next paragraph. Since it is also necessary to Since it is also necessary to accurately set the specimenaccurately set the specimen in th in the correct focal plane of the objective lens, a e correct focal plane of the objective lens, a z-axis or vertical z-axis or vertical movement is always providedmovement is always provided to allow accurate eucentric po to allow accurate eucentric positioning. sitioning. Modern side-entry stages offer Modern side-entry stages offer high resolution capabilitieshigh resolution capabilities ne nearly comparable to top-entry stages and permit more versatilarly comparable to top-entry stages and permit more versatility for specimen manipulation and orientation for analytical ity for specimen manipulation and orientation for analytical purposes. purposes. For these reasons, the side-entry stage is currently favored ovFor these reasons, the side-entry stage is currently favored over the top-entry stage in the latest generation of TEMs.er the top-entry stage in the latest generation of TEMs.

7272

Special StagesSpecial Stages It is possible to It is possible to manipulate the specimenmanipulate the specimen in the electron in the electron

microscope in a number of ways using special specimen microscope in a number of ways using special specimen stages or holders. stages or holders.

For instance, the specimen may be subjected to For instance, the specimen may be subjected to stretching and compression in a stretching and compression in a tensile stagetensile stage,, and and heating or cooling in specially modified heating or cooling in specially modified thermal stagesthermal stages..

Of particular interest to biologists is the Of particular interest to biologists is the cold stagecold stage,, since since it permits the examination of it permits the examination of rapidly frozen specimensrapidly frozen specimens (such as (such as live virus preparations)live virus preparations) that are still that are still hydrated hydrated and have not been exposed to chemical fixation or and have not been exposed to chemical fixation or stainingstaining. .

Besides examination of fluid specimens, it is also possible Besides examination of fluid specimens, it is also possible to study to study ultrathin frozen, hydrated sections of ultrathin frozen, hydrated sections of unprocessed biological materials for elemental analysisunprocessed biological materials for elemental analysis. .

Although specimen preparatory techniques are still being Although specimen preparatory techniques are still being refined, refined, cold stages offer tremendous potentialcold stages offer tremendous potential when when combined with the analytical capabilities of the TEM.combined with the analytical capabilities of the TEM.

7373

Imaging SystemImaging SystemThis part of the microscope includes This part of the microscope includes

the the objective, intermediate, and objective, intermediate, and projector lensesprojector lenses. .

It is involved in the It is involved in the generation of the generation of the imageimage and and the magnification and the magnification and projection of the final imageprojection of the final image onto a onto a viewing screenviewing screen or or camera systemcamera system of of the microscope. the microscope.

7474

7575

Objective Lens.Objective Lens. By far, this is the By far, this is the single most important lenssingle most important lens in the in the trantransmission electron microscopesmission electron microscope, since it forms the initial i, since it forms the initial image that is further magnified by the other imaging lenmage that is further magnified by the other imaging lenses. ses. In order to achieve such In order to achieve such high resolutionshigh resolutions, the lens must , the lens must be highly energized to obtain the short, be highly energized to obtain the short, 1 to 2 mm focal l1 to 2 mm focal lengths necessaryengths necessary. . The lens must be The lens must be free of astigmatismfree of astigmatism and have and have minimal minimal aberrations. aberrations. This means that theThis means that the polepieces polepieces must be constructed fr must be constructed from om homogeneously blended metalshomogeneously blended metals, be , be as symmetrical as symmetrical as possibleas possible, and contain devices, or , and contain devices, or stigmatorsstigmators, for corr, for correcting astigmatism. ecting astigmatism. The objective lens is used primarily to The objective lens is used primarily to focus the image.focus the image.

7676

Objective LensObjective Lens The objective lens also The objective lens also initially magnifiesinitially magnifies the image the image

whereas other lenses are used to magnify the image whereas other lenses are used to magnify the image further. further.

Of all of the lenses used in the magnification of an Of all of the lenses used in the magnification of an image, the image, the objective lens is the least variableobjective lens is the least variable so that so that it can maintain the it can maintain the very short focal lengths necessary very short focal lengths necessary for high resolutionfor high resolution and still be convenient to focus and still be convenient to focus (i.e., if the strength of the objective lens were varied (i.e., if the strength of the objective lens were varied over a wide range, refocusing would require major over a wide range, refocusing would require major adjustments of the lens current to the lens). adjustments of the lens current to the lens).

Currently, as Currently, as magnifications are changedmagnifications are changed, the , the adjustments to the objective lensadjustments to the objective lens needed to bring needed to bring the the image into focusimage into focus are not excessive. are not excessive.

7777

Objective Lens.Objective Lens. Because any Because any fluctuations in either lensfluctuations in either lens current current or high voltage would affect the or high voltage would affect the focus of the ofocus of the objective lensbjective lens, both must be made extremely st, both must be made extremely stable. able. Since Since contaminationcontamination may introduce astigmati may introduce astigmatism into sm into any lens systemany lens system, some way of minimizi, some way of minimizing contamination in the objective lens is needng contamination in the objective lens is needed. ed. Such devices, called Such devices, called anticontaminatorsanticontaminators,, are n are now essential for high quality work. ow essential for high quality work.

7878

Objective Lens.Objective Lens. Images are formed in the objective lens by a "Images are formed in the objective lens by a "ssubtractiveubtractive" action. " action. Depending on Depending on specimen thickness and densityspecimen thickness and density of of various parts of the specimenvarious parts of the specimen, some , some electroelectrons (inelastically scattered ones) will pass throuns (inelastically scattered ones) will pass through the specimengh the specimen and into and into subsequent lensessubsequent lenses w with a loss of some energy. ith a loss of some energy. Other electrons may be deflectedOther electrons may be deflected upon contac upon contact with t with parts of the specimenparts of the specimen and and renderedrendered unaunableble to enter the to enter the objective and other imaging leobjective and other imaging lensesnses. .

7979

Objective Lens.Objective Lens. Still other electrons may Still other electrons may lose all of their lose all of their

energyenergy upon impact with the specimen upon impact with the specimen and are likewise lost. and are likewise lost.

Most of the electronsMost of the electrons that enter the that enter the objective lens are ultimately objective lens are ultimately projected projected onto the phosphorescentonto the phosphorescent viewing screen viewing screen to cause a to cause a certain level of brightnesscertain level of brightness. .

The The more electronsmore electrons passing through passing through any any one pointone point on the specimen, the on the specimen, the brighter brighter the imagethe image generated. generated.

8080

Objective Lens.Objective Lens. Regardless of how an Regardless of how an electron is lostelectron is lost, this loss is evide, this loss is evidenced on the nced on the screen as a darker regionscreen as a darker region. . Hence, areas of Hence, areas of high density/thicknesshigh density/thickness will appear will appear dardarkerker than areas of than areas of less density/thicknessless density/thickness resulting in va resulting in various "rious "gray levelsgray levels" on the screen. " on the screen. Since Since biological specimensbiological specimens have inherently have inherently small densmall density differencessity differences between the between the various parts of the cellsvarious parts of the cells, , it is necessary to it is necessary to enhance these differences by reactinenhance these differences by reacting high density metalsg high density metals (osmium, lead, uranium, etc.) wi (osmium, lead, uranium, etc.) with specific subcellular structures. th specific subcellular structures. These heavy metals are introduced during the specimThese heavy metals are introduced during the specimen preparation processes of fixation and staining.en preparation processes of fixation and staining.

8181

Objective Lens.Objective Lens. Apertures in Objective LensApertures in Objective Lens.. As will be illustrated in s As will be illustrated in subsequent chapters, obtaining a thin specimen with gubsequent chapters, obtaining a thin specimen with good contrast is not always easily done. ood contrast is not always easily done. The The function of the objective aperture is primarily to efunction of the objective aperture is primarily to enhance contrastnhance contrast by trapping more of the by trapping more of the peripherally peripherally deflected electronsdeflected electrons (Figure 6.33). (Figure 6.33). Apertures of various sizes may be positioned in the Apertures of various sizes may be positioned in the popolepiece gaplepiece gap in the in the back focal planeback focal plane just under the spec just under the specimen. imen. Arranged on a similar Arranged on a similar positioning rodpositioning rod as the condense as the condenser apertures, these apertures are much smaller in size r apertures, these apertures are much smaller in size ((70, 50, 30, and 20 70, 50, 30, and 20 μμmm, for example) and more prone t, for example) and more prone to contamination. o contamination. SmallSmall objective apertures objective apertures give give increased contrastincreased contrast, alt, although at the expense of hough at the expense of overall overall illumination and resolillumination and resolution.ution.

8282

Figure 6.33Objective aperture located between upper and lower parts of polepiece, just under the specimen. The major function of the aperture is to help remove peripherally deflected electrons to enhance image contrast. In addition to the specimen and objective aperture, a chilled anticontaminator blade (see Figure 6.34) may also be inserted just above the specimen (or sometimes above and below the specimen) to prevent contaminants from condensing on specimen.

8383

Objective Lens.Objective Lens. Photographers make Photographers make use of aperturesuse of apertures not only to not only to

control the amount of illuminationcontrol the amount of illumination entering a lens, entering a lens, but further to control the but further to control the depth of fielddepth of field. .

It is well known that "It is well known that "stopping downstopping down" or " or decreasing the size of the aperturedecreasing the size of the aperture results in results in bringing more of the bringing more of the foreground and background foreground and background into focusinto focus, whereas , whereas wide open apertureswide open apertures result in result in only only a narrow zonea narrow zone being in focus. being in focus.

Depth of fieldDepth of field, therefore, refers to the , therefore, refers to the depth in depth in the specimen planethe specimen plane that is in focus. that is in focus. As is As is demonstrated in Equation 6.8, demonstrated in Equation 6.8, smaller apertures smaller apertures increaseincrease the the depthdepth in the specimen that is in in the specimen that is in focus.focus.

8484

Objective Lens.Objective Lens.Equation 6.8:Equation 6.8: Depth of FieldDepth of Field

where where = wavelength of radiation = wavelength of radiation = aperture angle= aperture angle

8585

Objective Lens.Objective Lens. If we are using an accelerating voltage of 60 kV,If we are using an accelerating voltage of 60 kV, the wavelength of the electron is 0.005 nm. the wavelength of the electron is 0.005 nm. A large A large 200 μm aperture200 μm aperture would generate an ap would generate an aperture angle of illumination in the objective leerture angle of illumination in the objective lens of approximately ns of approximately 1010-2-2 radians radians. . Upon substitution in the equation, we obtain a Upon substitution in the equation, we obtain a depth of field of approximately depth of field of approximately 50 nm50 nm. . Now, if a Now, if a 100 μm aperture100 μm aperture is used, the aperture is used, the aperture angle is approximately angle is approximately 1010-3-3 radians radians, which yiel, which yields a ds a 100 times100 times greater depth of field of greater depth of field of 5 μm.5 μm.

8686

Objective Lens.Objective Lens. Consequently, when using Consequently, when using smaller aperturessmaller apertures

in both the in both the objective and condenser lensesobjective and condenser lenses to to generate generate narrow aperture anglesnarrow aperture angles, the , the entire entire depth of the specimen is in focusdepth of the specimen is in focus. .

This is in This is in contrastcontrast to the light microscope, to the light microscope, where where larger aperture angles result in rather larger aperture angles result in rather narrow depths of fieldnarrow depths of field, making it necessary to , making it necessary to focus through the various levels to view the focus through the various levels to view the entire depth in the specimen. entire depth in the specimen.

Depth of field and depth of focus are Depth of field and depth of focus are illustrated in Figure 6.36, later in this chapter. illustrated in Figure 6.36, later in this chapter.

8787

Figure 6.36Depth of field (Dfi) occurs in the object plane, Depth of focus (Dfo) refers to the depth in the image plane that is in focus. In the bottom figure, note that an aperture increases both the depth of field and depth of focus.

8888

Objective Lens.Objective Lens. AperturesApertures are usually constructed from are usually constructed from high high

melting point metalsmelting point metals such as molybdenum or such as molybdenum or platinum. platinum.

Although they may be configured as single discs Although they may be configured as single discs with a central hole, they are more commonly with a central hole, they are more commonly fabricated from thin foils cut into a strip. fabricated from thin foils cut into a strip.

Individual holes are precisely drilled through the Individual holes are precisely drilled through the metal and scrutinized for burrs that would affect metal and scrutinized for burrs that would affect the symmetry of the field. the symmetry of the field.

Using the external controls on the TEM column, a Using the external controls on the TEM column, a single hole may be selected and positioned single hole may be selected and positioned symmetrically around the axis of the electron symmetrically around the axis of the electron beam. beam.

8989

Objective Lens.Objective Lens. It is possible to purchase standard strips It is possible to purchase standard strips

containing holes commonly used in a particular containing holes commonly used in a particular instrument, or one can order customized aperture instrument, or one can order customized aperture strips with specific types and thickness of metal strips with specific types and thickness of metal and specified hole diameters. and specified hole diameters.

For optimum performance, apertures must be For optimum performance, apertures must be cleaned cleaned periodically, the periodically, the frequency depending onfrequency depending on the types of the types of specimensspecimens and general cleanliness of and general cleanliness of the microscope itself. the microscope itself.

MolybdenumMolybdenum apertures are cleaned by placing apertures are cleaned by placing the strip into a the strip into a flat holderflat holder of tungsten or of tungsten or molybdenum and molybdenum and heating the strip in a vacuum heating the strip in a vacuum evaporatorevaporator (to avoid oxidation). (to avoid oxidation).

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Objective Lens.Objective Lens. After reaching a cherry red color, most After reaching a cherry red color, most

contamination will be evaporated and contamination will be evaporated and removed by the vacuum system. removed by the vacuum system.

However, it may be necessary to repeat this However, it may be necessary to repeat this process several times. process several times.

One should not exceed the cherry red color, One should not exceed the cherry red color, since the molybdenum may since the molybdenum may weld ontoweld onto the the holder. holder.

Platinum strips may be cleaned by passage Platinum strips may be cleaned by passage through a propane gas flame followed by through a propane gas flame followed by immersion in hydrofluoric acid and ammonium immersion in hydrofluoric acid and ammonium hydroxide. Aperture strips are easier to clean hydroxide. Aperture strips are easier to clean if contamination has not built up over time. if contamination has not built up over time.

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Objective Lens.Objective Lens. A A thin foil aperturethin foil aperture may also be used in the may also be used in the

objective or condenser lenses. Such apertures objective or condenser lenses. Such apertures are made of an extremely thin layer of gold are made of an extremely thin layer of gold and are "self-cleaning." and are "self-cleaning."

In practice, one must regularly run the focused In practice, one must regularly run the focused electron beam over the rim of the aperture in electron beam over the rim of the aperture in order to bake off the contaminants. order to bake off the contaminants.

When such foils can no longer be cleaned When such foils can no longer be cleaned using such a procedure, they must be using such a procedure, they must be replaced.replaced.

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Anticontaminators in Specimen AreaAnticontaminators in Specimen Area AnticontaminatorsAnticontaminators are found in close proximit are found in close proximity to the specimen, aperture, and polepiece of ty to the specimen, aperture, and polepiece of the objective lens. he objective lens. They are essentially They are essentially metal surfacesmetal surfaces that are that are chichilled with liquid nitrogenlled with liquid nitrogen from a reservoir outsi from a reservoir outside the column of the microscope. de the column of the microscope. Most contaminants originating from the speciMost contaminants originating from the specimen or the microscope will men or the microscope will condense ontocondense onto the the extremely extremely cold anticontaminatorcold anticontaminator and be remo and be removed from the system. ved from the system.

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Anticontaminators in Specimen AreaAnticontaminators in Specimen Area Anticontaminators are sometimes called Anticontaminators are sometimes called cold fingerscold fingers.. Some anticontaminators may resemble an aperture hSome anticontaminators may resemble an aperture holder, except that the brass plate is much thicker and older, except that the brass plate is much thicker and the aperture much larger (Figure 6.34). the aperture much larger (Figure 6.34). Other anticontaminators are Other anticontaminators are ring-shaped and encircle ring-shaped and encircle the specimenthe specimen. . Since anticontaminators must fit into the same crampSince anticontaminators must fit into the same cramped space that also accommodates the specimen and oed space that also accommodates the specimen and objective aperture, they must be designed very carefullbjective aperture, they must be designed very carefully. y. Anticontaminators Anticontaminators must be polished clean periodicallmust be polished clean periodically to remove condensed materials and then carefully py to remove condensed materials and then carefully positioned to avoid contact with the specimen holder oositioned to avoid contact with the specimen holder or objective aperture r objective aperture

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Figure 6.34Specimen anticontaminator or cold finger. The large container (c) is filled with liquid nitrogen to chill the cold finger blade (b) that is located just above and below the specimen. An O-ring seals the apparatus from the atmosphere.

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Astigmatism CorrectionAstigmatism Correction StigmatorsStigmators are located beneath not only the are located beneath not only the objectivobjective e but also the but also the condenser and intermediate lensescondenser and intermediate lenses. . They function to correct the They function to correct the radial lens asymmetriesradial lens asymmetries t that hat preventprevent one one from focusing the image in all directiofrom focusing the image in all directionsns and and generating circular illumination spotsgenerating circular illumination spots. . Since an astigmatic lens is stronger in one direction (nSince an astigmatic lens is stronger in one direction (north-south, for instance) than another, one orth-south, for instance) than another, one creates a ccreates a compensating field of equivalent strengthompensating field of equivalent strength in the opposi in the opposite direction (east-west). te direction (east-west). Two parameters must be considered: Two parameters must be considered: direction of the direction of the astigmatismastigmatism ( (azimuth)azimuth) and and strength of the astigmatisstrength of the astigmatismm ( (amplitude).amplitude). One must be able to adjust both variables to suit the pOne must be able to adjust both variables to suit the particular situation. articular situation.

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Astigmatism CorrectionAstigmatism Correction Older stigmators were composed of Older stigmators were composed of pairs of magnetic pairs of magnetic slugsslugs that could be that could be mechanically rotated into positionmechanically rotated into position to compensate for astigmatism. to compensate for astigmatism. Newer microscopes use primarily Newer microscopes use primarily electromagnetic stielectromagnetic stigmatorsgmators since they are less expensive to build, easier t since they are less expensive to build, easier to use, and somewhat more precise in their correction. o use, and somewhat more precise in their correction. Electromagnetic stigmatorsElectromagnetic stigmators may consist of eight tiny e may consist of eight tiny electromagnets encircling the lens field. lectromagnets encircling the lens field. By By varying the strength and polarity of variousvarying the strength and polarity of various sets of sets of magnets, one can control both amplitude and azimutmagnets, one can control both amplitude and azimuth in order to generate h in order to generate a symmetrical magnetic fielda symmetrical magnetic field (Figure 6.35). (Figure 6.35). When stigmators become dirty, they will no longer When stigmators become dirty, they will no longer effeffectively compensate for astigmatismectively compensate for astigmatism and must be wit and must be withdrawn from the microscope and cleaned.hdrawn from the microscope and cleaned.

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Figure 6.35(A) Conceptual drawing of electromagnetic stigmatorshowing orientation of eight electromagnets around thelens axis. Strength and direction are controlled byadjusting appropriate combinations of magnets to generate a symmetrical field. The stigmator is locatedunder the condenser and the objective lens polepieces.(B) Actual stigmator apparatus taken from an electron microscope. The large arrow indicates one of the eightelectromagnetic iron slugs oriented around the centralaxis. The entire apparatus fits up into the bore of theobjective lens so that the area indicated in the largearrow is positioned just under the specimen. The smaller arrow points out individual electrical contacts through which current flows to energize the electromagnets. The close-up photograph (bottom) shows some of the electromagnets that are positioned near the specimen (arrow).

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Intermediate (Diffraction) Intermediate (Diffraction) LensLens

Intermediate (Diffraction) LensIntermediate (Diffraction) Lens.. As one As one proceeds down the column, this lens proceeds down the column, this lens immediately follows and is constructed immediately follows and is constructed similarly to the objective lenssimilarly to the objective lens. .

In older, simpler microscopes, In older, simpler microscopes, magnification magnification is altered by varying the current to this is altered by varying the current to this lenslens, while in , while in newernewer microscopes the microscopes the preferred method is to use preferred method is to use combinations of combinations of several lenses toseveral lenses to allow a wider, allow a wider, distortion-distortion-free magnification rangefree magnification range. .

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Intermediate (Diffraction) Intermediate (Diffraction) LensLens

The major function of this lens is to The major function of this lens is to assist in the assist in the magnificationmagnification of the image from the objective lens. of the image from the objective lens.

At very At very low magnificationslow magnifications, the , the objective lens is objective lens is shut offshut off and the intermediate lens used in its place and the intermediate lens used in its place to to generate the primary image. generate the primary image.

Although the image produced by the Although the image produced by the very long very long focal length intermediate lensfocal length intermediate lens is is poorpoor compared to compared to that generated by using all three lenses, it is that generated by using all three lenses, it is adequate for low magnification work. adequate for low magnification work.

The intermediate lens may be equipped with an The intermediate lens may be equipped with an aperture that is used when operating the aperture that is used when operating the microscope in the diffraction mode (Chapter 15). microscope in the diffraction mode (Chapter 15).

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Projector LensProjector Lens Most modern transmission electron microscopes have Most modern transmission electron microscopes have

two projector lensestwo projector lenses (P1 and P2) that follow the (P1 and P2) that follow the intermediate lens. intermediate lens.

Both P1 and P2 are used to Both P1 and P2 are used to further magnify imagesfurther magnify images from the intermediate or diffraction lens. from the intermediate or diffraction lens.

Except for very Except for very high magnificationshigh magnifications, only , only three of the three of the fourfour imaging lenses are normally energized at any one imaging lenses are normally energized at any one time, and various time, and various triplet combinationstriplet combinations are used to are used to achieve the magnification range desired. achieve the magnification range desired.

In a microscope with In a microscope with four imaging lensesfour imaging lenses, the first , the first projector lens can also be used as a diffraction lens, projector lens can also be used as a diffraction lens, and it may be possible to and it may be possible to insert a specimeninsert a specimen into a into a specially specially modified holdermodified holder located either between P1 and located either between P1 and P2 or below P2 for specialized, low angle diffraction P2 or below P2 for specialized, low angle diffraction studies. studies.

As with As with intermediate lensesintermediate lenses, projector lenses suffer , projector lenses suffer from from distortionsdistortions that have that have less effect on resolutionless effect on resolution than than do do aberrations occurringaberrations occurring in the objective lens. in the objective lens.

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Projector LensProjector Lens Projector lenses are said to have Projector lenses are said to have great great

depth of focusdepth of focus,, meaning that the meaning that the final final image remainsimage remains in focus for a long distance in focus for a long distance along the optical axis. This is determined by along the optical axis. This is determined by Equation 6.9 Equation 6.9

Equation 6.9: Depth of FocusEquation 6.9: Depth of Focus

where: M = total magnification RP = resolving power of instrument being used = aperture angle established by objective lens

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Projector LensProjector Lens At a magnification of 100,000×, in an instrument with At a magnification of 100,000×, in an instrument with

resolving power of resolving power of 0.2 nm0.2 nm and having an aperture and having an aperture angle of angle of 1010-2-2 radians radians, the depth of focus of the , the depth of focus of the projector lens may be calculated to be projector lens may be calculated to be 200 meters200 meters. .

This becomes important when one realizes that the This becomes important when one realizes that the photographic film is not photographic film is not in the same planein the same plane as the as the viewing screenviewing screen. .

For the same reason, it is possible to For the same reason, it is possible to locate multiple locate multiple image recordingimage recording devices at various points beyond the devices at various points beyond the projector lensprojector lens, since they will all be in focus. , since they will all be in focus.

However, the However, the magnification will increasemagnification will increase as one as one moves moves farther awayfarther away from the projector lens. from the projector lens.

The relationship between depth of field and depth of The relationship between depth of field and depth of focus relative to aperture angle is shown in Figure focus relative to aperture angle is shown in Figure 6.36. 6.36.

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Figure 6.36Depth of field (Dfi) occurs in the object plane, while depth of focus (Dfo) refers to the depth in the image plane that is in focus. In the bottom figure, note that an apertureincreases both the depth of field and depth of focus.

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Viewing System and Viewing System and CameraCamera

The final image is projected onto a The final image is projected onto a viewing screenviewing screen coa coated with a ted with a phosphorescent zinc-activated cadmium suphosphorescent zinc-activated cadmium sulfide powderlfide powder attached to the screen with a binder suc attached to the screen with a binder such as h as cellulose nitratecellulose nitrate. . Most electron microscopes provide for an inclination Most electron microscopes provide for an inclination of the viewing screen so that the image may be conveof the viewing screen so that the image may be conveniently examined either with the unaided eye or with niently examined either with the unaided eye or with a a stereomicroscopestereomicroscope called the binoculars. called the binoculars. With the With the stereomicroscopstereomicroscope, although the image may ae, although the image may appear to be rough due to the ppear to be rough due to the 100 μm-sized100 μm-sized grains of p grains of phosphorescent particles making up the screen, it is nehosphorescent particles making up the screen, it is necessary to view a magnified image in order to focus accessary to view a magnified image in order to focus accurately. curately.

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A A shutter shutter is provided to time the is provided to time the exposure so exposure so that the proper negative densitythat the proper negative density (as (as determined by the previous calibration) may determined by the previous calibration) may be obtained. be obtained.

Most Most electron microscopeselectron microscopes have timers that have timers that vary from a fraction of a second to vary from a fraction of a second to "hold" "hold" positionspositions in which a timer may be used for in which a timer may be used for very long manual exposures. very long manual exposures.

Electron micrographs are exposed for Electron micrographs are exposed for 0.5 to 0.5 to 2 seconds2 seconds in order to in order to record all density levelsrecord all density levels and to and to minimize image shift or minimize image shift or driftdrift (i.e., slow (i.e., slow movement of the image after exposure to movement of the image after exposure to the beam). the beam).

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Once the time has been selected, the illumination Once the time has been selected, the illumination level is adjusted with the C1 and C2 lens controls level is adjusted with the C1 and C2 lens controls until the exposure meter reaches the calibration until the exposure meter reaches the calibration point. point.

The film is then advanced under the viewing The film is then advanced under the viewing screen, and the screen is moved to screen, and the screen is moved to permit permit electrons to pass onto the filmelectrons to pass onto the film. .

As one begins to As one begins to raise the viewing screenraise the viewing screen, the , the beam is blocked by the shutter until the beam is blocked by the shutter until the screen is screen is totally raisedtotally raised. .

The shutter is then opened for the proper interval, The shutter is then opened for the proper interval, after which the beam is again blocked until the after which the beam is again blocked until the screen is repositioned. screen is repositioned.

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Major Operational Modes Major Operational Modes of the Transmission of the Transmission Electron MicroscopeElectron Microscope

High ContrastHigh ContrastHigh ResolutionHigh Resolution

Dark FieldDark FieldDiffractionDiffraction

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Major Operational Modes of the Major Operational Modes of the Transmission Electron Transmission Electron

MicroscopeMicroscope During the alignment procedure, one During the alignment procedure, one

should be aware that the conventional should be aware that the conventional transmission electron microscope may be transmission electron microscope may be set up for operation in several different set up for operation in several different operational modes. operational modes.

Depending on the design of the Depending on the design of the microscope, this may involve relatively microscope, this may involve relatively few few or many mutually exclusive adjustments. or many mutually exclusive adjustments.

In addition, certain specimen preparation In addition, certain specimen preparation techniques may be utilized to further techniques may be utilized to further enhance these operational modes. enhance these operational modes.

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High ContrastHigh ContrastA constant problem with biological A constant problem with biological

specimens is their specimens is their low contrastlow contrast. . In the high contrast mode, the In the high contrast mode, the

instrument is adjusted to instrument is adjusted to give contrast give contrast at the expense of high resolutionat the expense of high resolution. .

As a result, this mode is generally used As a result, this mode is generally used at magnifications at magnifications under 50,000under 50,000 X. X.

The conditions that may be changed to The conditions that may be changed to enhance contrast are summarized below enhance contrast are summarized below

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How to Obtain High How to Obtain High ContrastContrast

1. 1. The focal length of the objective lens The focal length of the objective lens is increasedis increased..

This necessitates using shorter specimen This necessitates using shorter specimen holder cartridges (Figure 6.30, left) in a top holder cartridges (Figure 6.30, left) in a top entry stage to position the specimen higher entry stage to position the specimen higher in the objective lens. in the objective lens.

In a side entry stage, adjustment of the z-axis In a side entry stage, adjustment of the z-axis or specimen positioning may also be needed or specimen positioning may also be needed if a special holder is not provided. if a special holder is not provided.

It may be recalled that longer focal lengths It may be recalled that longer focal lengths result result in narrower aperture angles, a in narrower aperture angles, a worsening of chromatic aberration, and a loss worsening of chromatic aberration, and a loss of resolutionof resolution. .

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Figure 6.30(left) Short, top-entry grid holder for high contrast,low-magnification work. Resolution is not as good withthis type of grid holder since the specimen is placedhigher in the objective lens, necessitating a longer focallength of the lens. (right) Standard top-entry specimengrid holder for high resolution work. The specimen gridis placed on the end of the grid holder shaft and held inplace with a sleeve that is slipped over the shaft (arrow).These holders are placed in the specimen stage with thegrids in the downward position in the polepiece.

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2. 2. Lower accelerating voltages are usedLower accelerating voltages are used.. The The resulting lower energy electrons are resulting lower energy electrons are more readily more readily affectedaffected by differences in by differences in specimen density and specimen density and thicknessthickness, and contrast will be thereby increased. , and contrast will be thereby increased.

Unfortunately, this interaction with the specimen Unfortunately, this interaction with the specimen generates a population of generates a population of imaging electrons with imaging electrons with a wide range of energiesa wide range of energies, resulting in an , resulting in an increase increase in chromatic aberration.in chromatic aberration.

Lower accelerating voltages are also Lower accelerating voltages are also more more damaging to the specimendamaging to the specimen, since the electrons , since the electrons are slowed down more and are slowed down more and transfer more energy transfer more energy to the specimento the specimen, resulting in excessive heating. , resulting in excessive heating.

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How to Obtain High Contrast How to Obtain High Contrast 2. 2. Lower accelerating voltages are Lower accelerating voltages are

usedused Lower energy electrons are more Lower energy electrons are more susceptiblesusceptible

to to poor vacuum conditionspoor vacuum conditions, with the , with the exacerbation of exacerbation of chromatic aberrationchromatic aberration. .

Clean, high vacuums are needed to minimize Clean, high vacuums are needed to minimize electron energy losses, and the microscope electron energy losses, and the microscope itself should be clean, since these electrons itself should be clean, since these electrons are more easily affected by astigmatism. are more easily affected by astigmatism.

Lastly, it will be recalled that Lastly, it will be recalled that lower energy lower energy electrons have longer wavelengthselectrons have longer wavelengths, so that , so that the resolving power will be degraded. the resolving power will be degraded.

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3. 3. Smaller objective apertures should be utilSmaller objective apertures should be utilizedized.. These apertures will These apertures will remove more of the peripremove more of the peripherally deflected electronsherally deflected electrons from the specimen, from the specimen, so that the subtractive image from the objectiso that the subtractive image from the objective lens will be accentuated in contrast (i.e., thve lens will be accentuated in contrast (i.e., the signal-to-noise ratio is increased). e signal-to-noise ratio is increased). Small apertures are more prone to astigmatisSmall apertures are more prone to astigmatism problems, making clean vacuums and specim problems, making clean vacuums and specimen anticontaminators essential. men anticontaminators essential.

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4. 4. Photographic procedures may be employedPhotographic procedures may be employed.. Most images generated in the Most images generated in the transmission electron transmission electron

microscopemicroscope are enhanced for contrast using are enhanced for contrast using photographic techniques. photographic techniques.

During exposure of the electron micrograph, the During exposure of the electron micrograph, the sensitivity of the exposure meter may be adjusted to sensitivity of the exposure meter may be adjusted to slightly overexpose the film. slightly overexpose the film.

Underdevelopment will then enhance the contrast Underdevelopment will then enhance the contrast rangerange in the final negative. in the final negative.

Details will necessarily be lost in the intermediate Details will necessarily be lost in the intermediate density ranges. Of course, during the printing of the density ranges. Of course, during the printing of the negative, one may use higher contrast photographic negative, one may use higher contrast photographic papers (see Chapter 8). papers (see Chapter 8).

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5. 5. The specimen may be prepared to enhance contrThe specimen may be prepared to enhance contrast.ast. Standard fixation and staining techniques will increasStandard fixation and staining techniques will increase density by depositing the e density by depositing the heavy metalsheavy metals along variou along various organelles. s organelles. Certain Certain embedding media (polyethelene glycol)embedding media (polyethelene glycol) that that may be dissolved or etched away will help boost contrmay be dissolved or etched away will help boost contrast, or one may utilize stained, frozen sections withouast, or one may utilize stained, frozen sections without any embedding media. t any embedding media. The easiest approach is simply to The easiest approach is simply to cut thicker sectionscut thicker sections; ; however, the resulting however, the resulting chromatic aberrationchromatic aberration and supe and superimposition of structure will degrade resolution. rimposition of structure will degrade resolution.

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High ResolutionHigh Resolution Most of the conditions used to achieve Most of the conditions used to achieve

high resolutionhigh resolution in the in the electron microscopeelectron microscope are the are the opposite conditionsopposite conditions discussed discussed above for the high contrast mode. above for the high contrast mode.

Since Since contrastcontrast will be lacking in these will be lacking in these specimens, efforts should be made to specimens, efforts should be made to boost contrast using appropriate boost contrast using appropriate specimen preparation and darkroom specimen preparation and darkroom techniquestechniques, as described in the previous , as described in the previous section. section.

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How to Obtain High How to Obtain High ResolutionResolution

1. 1. The objective lens should be adjusted to gThe objective lens should be adjusted to give the ive the shortest possible focal lengthshortest possible focal length and th and the proper specimen holders used. e proper specimen holders used. In some systems, this is simply a matter of preIn some systems, this is simply a matter of pressing a single button, whereas, in certain micrssing a single button, whereas, in certain microscopes several lens currents must be changeoscopes several lens currents must be changed concomitantly. d concomitantly. Perhaps it may even be necessary to insert a diPerhaps it may even be necessary to insert a different polepiece in the objective lens. fferent polepiece in the objective lens.

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2. 2. Adjustments to the gun, such as the Adjustments to the gun, such as the use of higher use of higher accelerating voltages,accelerating voltages, will result in higher resolution will result in higher resolution for the reasons already mentioned in the discussion ofor the reasons already mentioned in the discussion on high contrast. n high contrast. Chromatic aberrationChromatic aberration may be further lessened by usin may be further lessened by using g field emission gunsfield emission guns since the energy spread of electr since the energy spread of electrons generated from such guns is considerably narrowons generated from such guns is considerably narrower. (The energy spread for tungsten = 2 eV while field eer. (The energy spread for tungsten = 2 eV while field emission = 0.2–0.5 eV.) mission = 0.2–0.5 eV.) In an electron microscope equipped with a conventioIn an electron microscope equipped with a conventional gun, a pointed tungsten filament will generate a mnal gun, a pointed tungsten filament will generate a more coherent, point source of electrons with better resore coherent, point source of electrons with better resolution capabilities. olution capabilities.

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3. 3. Use apertures of appropriate size.Use apertures of appropriate size. For most specimens, For most specimens, larger objective lens apertureslarger objective lens apertures

should be used to minimize diffraction effects. should be used to minimize diffraction effects. If contrast is too low due to the larger objective If contrast is too low due to the larger objective

aperture, aperture, smaller apertures may be usedsmaller apertures may be used but resolution but resolution will be diminished. will be diminished.

In addition, they must be kept clean since In addition, they must be kept clean since dirtdirt will have will have a more pronounced effect on a more pronounced effect on astigmatismastigmatism. .

Small condenser lens apertures will diminish spherical Small condenser lens apertures will diminish spherical aberration, but this will be at the expense of aberration, but this will be at the expense of overall overall illuminationillumination. .

The illumination levels may be improved by altering the The illumination levels may be improved by altering the bias to effect greater gun emissions; however, this may bias to effect greater gun emissions; however, this may thermally damage the specimen. thermally damage the specimen.

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4. 4. Specimen preparation techniquesSpecimen preparation techniques may also enhance the resolution capability. may also enhance the resolution capability.

Extremely thin sections, for instance, will Extremely thin sections, for instance, will diminish chromatic aberration. diminish chromatic aberration.

Whenever possible, no supporting Whenever possible, no supporting substrates should be used on the grid. substrates should be used on the grid.

To achieve adequate support, this may To achieve adequate support, this may require the use of holey films with a larger require the use of holey films with a larger than normal number of holes (holey nets, than normal number of holes (holey nets, see Chapter 4). see Chapter 4).

The areas viewed are limited to those over The areas viewed are limited to those over the holes. the holes.

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5. 5. Miscellaneous conditionsMiscellaneous conditions such as such as shorter viewing shorter viewing and exposure timesand exposure times will minimize contamination, drift, will minimize contamination, drift, and specimen damage, and help to preserve fine stru and specimen damage, and help to preserve fine structural details. ctural details. Some of the newest microscopes have special accessoSome of the newest microscopes have special accessories for minimal electron dose observation of the specries for minimal electron dose observation of the specimen and may even utilize electronic image intensifierimen and may even utilize electronic image intensifiers to enhance the brightness and contrast of the image. s to enhance the brightness and contrast of the image. Anticontaminators over the diffusion pumps and speciAnticontaminators over the diffusion pumps and specimen area will diminish contamination and resolution lmen area will diminish contamination and resolution loss. oss. High magnifications will be necessary, so careful adjuHigh magnifications will be necessary, so careful adjustment of the illuminating system is important. stment of the illuminating system is important.

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It may take nearly an hour for the eyes to totally adapt It may take nearly an hour for the eyes to totally adapt to the low light levels, and this adaption will be lost if to the low light levels, and this adaption will be lost if one must leave the microscope room. one must leave the microscope room. Alignment must be well done and Alignment must be well done and stigmation must be stigmation must be checked periodicallychecked periodically during the viewing session. during the viewing session. The circuitry of the microscope should be stabilized bThe circuitry of the microscope should be stabilized by allowing the lens currents and high voltage to warm y allowing the lens currents and high voltage to warm up for 1 to 2 hours before use. up for 1 to 2 hours before use. Bent specimen grids should be avoidedBent specimen grids should be avoided since they ma since they may place the specimen in an improper focal plane for opy place the specimen in an improper focal plane for optimum resolution. timum resolution. In addition, they prevent accurate magnification deterIn addition, they prevent accurate magnification determination and are more prone to drift since the suppormination and are more prone to drift since the support films are often detached. t films are often detached.

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DarkfieldDarkfield In the normal operating mode of the transmission eleIn the normal operating mode of the transmission electron microscope, the unscattered rays of the beam arctron microscope, the unscattered rays of the beam are combined with some of the deflected electrons to foe combined with some of the deflected electrons to form a brightfield image. rm a brightfield image. As more of the As more of the deflected or scattered electronsdeflected or scattered electrons are eli are eliminated using smaller objective lens apertures, contrminated using smaller objective lens apertures, contrast will increase. ast will increase. If one moves the If one moves the objective aperture off axisobjective aperture off axis, as shown , as shown in Figure 6.50, left, in Figure 6.50, left, the unscattered electrons are now the unscattered electrons are now eliminated while more of the scattered electrons enteeliminated while more of the scattered electrons enter the aperture. r the aperture. This is a crude form of darkfield illumination. This is a crude form of darkfield illumination. Unfortunately, the off-axis electrons have more aberraUnfortunately, the off-axis electrons have more aberrations and the image is of poor quality. tions and the image is of poor quality.

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Figure 6.50Schematic diagram showing two ways of setting upmicroscope for darkfield imaging: (left) displacementof objective aperture off-axis; (right) tilt of illuminationsystem into on-axis objective aperture.

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DarkfieldDarkfield Higher resolution darkfield may be obtained by Higher resolution darkfield may be obtained by tilting tilting the illumination system so that the beam strikes the sthe illumination system so that the beam strikes the specimen at an angle. pecimen at an angle. If the objective aperture is left normally centered, it wiIf the objective aperture is left normally centered, it will now accept only the scattered, on-axis electrons anll now accept only the scattered, on-axis electrons and the image will be of high quality d the image will be of high quality (Figure 6.50, right).(Figure 6.50, right). Most microscopes now have a dual set of beam tilt coMost microscopes now have a dual set of beam tilt controls that will permit one to adjust the tilt for either bntrols that will permit one to adjust the tilt for either brightfield or darkfield operation. rightfield or darkfield operation. After alignment of the tilt for brightfield followed by a After alignment of the tilt for brightfield followed by a darkfield alignment, one may rapidly shift from one mdarkfield alignment, one may rapidly shift from one mode to the other with the flip of a switch. ode to the other with the flip of a switch. Both sets of controls also provide for separate stigmatBoth sets of controls also provide for separate stigmation controls to correct for any astigmatism introduced ion controls to correct for any astigmatism introduced by the tilting of the beam to large angles. by the tilting of the beam to large angles.

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DarkfieldDarkfieldThe darkfield mode can be used to The darkfield mode can be used to

enhance contrast in certain types of enhance contrast in certain types of unstained specimens (thin frozen unstained specimens (thin frozen sectionssections) or in ) or in negatively stainednegatively stained specimens. specimens.

An example of a darkfield image is An example of a darkfield image is shown in Figure 6.51. shown in Figure 6.51.

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Figure 6.51(top) Darkfield image obtained by tilting illumination system.(bottom) Same specimen viewed in standard brightfieldmode. Specimen consists of inorganic salt crystals.

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DiffractionDiffraction In specimens that contain crystals of In specimens that contain crystals of unknown compounknown compositionsition, the diffraction technique may be used to meas, the diffraction technique may be used to measure the spacing of the ure the spacing of the atomic crystalline lattice and deatomic crystalline lattice and determine the composition of thetermine the composition of the crystal, since different crystal, since different crystals have unique crystals have unique spacings of their latticesspacings of their lattices. . The The diffraction phenomenondiffraction phenomenon is based on the is based on the reflectioreflection or diffraction of the electron beam to certain angles n or diffraction of the electron beam to certain angles by a crystalline lattice.by a crystalline lattice. Instead of focusing a conventional image of the crystaInstead of focusing a conventional image of the crystal on the viewing screen using the objective lens, one ul on the viewing screen using the objective lens, one uses the ses the intermediate or diffraction lens to focus on the intermediate or diffraction lens to focus on the back focal planeback focal plane to see the to see the selected area diffraction (Sselected area diffraction (SAD) on the screen. AD) on the screen.

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DiffractionDiffraction Since the Since the crystalline lattice diffracts electrons crystalline lattice diffracts electrons to form bright spotsto form bright spots on the viewing screen (si on the viewing screen (similar to the mirrored rotating sphere sometimmilar to the mirrored rotating sphere sometimes used in ballrooms to reflect a light source oes used in ballrooms to reflect a light source onto the walls), the image will nto the walls), the image will consist of a centrconsist of a central, bright spot surrounded by a series of spots, al, bright spot surrounded by a series of spots, which are the reflections. which are the reflections. The central bright spot represents The central bright spot represents nondiffractnondiffracteded rays while the rays while the peripheral spots represent raperipheral spots represent rays diffracted at various angles.ys diffracted at various angles.

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DiffractionDiffraction The distance of these spots from the bright central spoThe distance of these spots from the bright central spot is t is inversely proportionalinversely proportional to the to the spacing of the crystallispacing of the crystalline lattice. ne lattice. A crystal with A crystal with small lattice spacingssmall lattice spacings will diffract the ele will diffract the electrons to ctrons to greater anglesgreater angles to give spots that are spaced f to give spots that are spaced far from the central spot. ar from the central spot. This is unfortunate for biologists, since This is unfortunate for biologists, since organic crystals,organic crystals, such as protein, with such as protein, with large lattice spacingslarge lattice spacings will diffract will diffract the beam so little that the spots will be crowded arounthe beam so little that the spots will be crowded around the central bright spotd the central bright spot and engulfed by its brilliance. and engulfed by its brilliance. With organic crystals, the specialized technique of high With organic crystals, the specialized technique of high dispersion electron diffractiondispersion electron diffraction must be used. must be used.

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Diffraction PracticesDiffraction Practices After the crystal is located (using the After the crystal is located (using the

standard bright-field imaging mode), the standard bright-field imaging mode), the crystal is centered on the viewing screen crystal is centered on the viewing screen and the and the objective aperture removed.objective aperture removed.

After placing the TEM in the After placing the TEM in the selected area selected area diffractiondiffraction (SAD) mode, an SAD aperture of (SAD) mode, an SAD aperture of the appropriate size is inserted to select the the appropriate size is inserted to select the area of the crystal one wishes to diffract. area of the crystal one wishes to diffract.

Focus sharply on the edge of the SAD Focus sharply on the edge of the SAD apertureaperture using the SAD (intermediate lens) using the SAD (intermediate lens) control. control.

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Diffraction PracticesDiffraction Practices Refocus the image using the objective lens focus Refocus the image using the objective lens focus

controls to bring the image into the same plane as controls to bring the image into the same plane as the intermediate aperture. (If contrast is the intermediate aperture. (If contrast is inadequate at this point, temporarily reinsert the inadequate at this point, temporarily reinsert the objective aperture to check focus and then remove objective aperture to check focus and then remove it before proceeding.) it before proceeding.)

Place the TEM into the diffraction mode (usually a Place the TEM into the diffraction mode (usually a button labeled ''D" or "DIFF") and ensure that the button labeled ''D" or "DIFF") and ensure that the second condenser (C2) lens is spread to prevent second condenser (C2) lens is spread to prevent burning of the viewing screen. burning of the viewing screen.

For photography, For photography, adjust the size of the diffraction adjust the size of the diffraction pattern using the camera length controlpattern using the camera length control, readjust , readjust the C2 lens so that the pattern is very the C2 lens so that the pattern is very dim,dim, and and focus the central bright spot as small as possiblefocus the central bright spot as small as possible using the intermediate lens. using the intermediate lens.

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Diffraction PracticesDiffraction Practices In order to cut down on the In order to cut down on the glare from the brightglare from the bright

central spot, a physical central spot, a physical beam stopper is inserted to beam stopper is inserted to covercover it. it.

Exposures are usually made for Exposures are usually made for 30 to 60 seconds30 to 60 seconds in in the manual mode since the illumination levels will be the manual mode since the illumination levels will be very low. very low.

Single crystals will generate separate spots while Single crystals will generate separate spots while polycrystalline specimens will produce so many spots polycrystalline specimens will produce so many spots around the central pointaround the central point that they will blend to form that they will blend to form a series of a series of concentric ringsconcentric rings (Figure 6.52). (Figure 6.52).

Some biological applications of diffraction may be to Some biological applications of diffraction may be to confirm that a crystal present in human lung tissue is confirm that a crystal present in human lung tissue is a form of asbestos, or to identify an unknown crystal a form of asbestos, or to identify an unknown crystal in a plant or bacterial cell. See also Chapter 15 and in a plant or bacterial cell. See also Chapter 15 and the reference sources at the end of this chapter. the reference sources at the end of this chapter.

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Figure 6.52Diffraction pattern obtained from polycrystallinespecimen showing characteristic ring pattern.

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Electrons, Waves, and Electrons, Waves, and ResolutionResolution

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Physicists have demonstrated that, besides being discPhysicists have demonstrated that, besides being discrete particles having a negative charge and a mass of rete particles having a negative charge and a mass of 9.1X109.1X10-23-23 kg, kg, electrons also have wave properties.electrons also have wave properties. In fact, the In fact, the wavelength (λ)wavelength (λ) of an electron is expressed of an electron is expressed by the equation of the French physicist by the equation of the French physicist de Brogliede Broglie as s as shown in Equation 6.3.hown in Equation 6.3. Equation 6.3: de Broglie Equation for Wavelength oEquation 6.3: de Broglie Equation for Wavelength of an Electronf an Electron λ=h/mv λ=h/mv

where h = Planck's constant (6.626 X 10where h = Planck's constant (6.626 X 10-23-23 ergs/sec) ergs/sec) m = mass of the electronm = mass of the electron v = electron velocityv = electron velocity

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After appropriate substitutions associating After appropriate substitutions associating kinetic energy to mass, velocity, and kinetic energy to mass, velocity, and accelerating voltage, the equation may be accelerating voltage, the equation may be expressed: expressed: λ=1.23/(V)λ=1.23/(V)1/21/2

Where: V = accelerating voltageWhere: V = accelerating voltage Therefore, if one were operating a Therefore, if one were operating a

transmission electron microscope at an transmission electron microscope at an accelerating voltage of accelerating voltage of 60 kV60 kV, the , the wavelength of the electron would be wavelength of the electron would be 0.005 0.005 nmnm, and the resolving power of the system—, and the resolving power of the system—after substitution of these values into after substitution of these values into Equation 6.2—should be approximately Equation 6.2—should be approximately 0.003 nm. 0.003 nm.

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Determine resolving powerDetermine resolving powerRadius of Airy Disc: the Radius of Airy Disc: the radius of the Airy radius of the Airy discdisc as measured to the first dark ring is as measured to the first dark ring is express by Equation 6.1:express by Equation 6.1:

r= 0.612 λ/n (sin α)r= 0.612 λ/n (sin α)

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In fact, the actual In fact, the actual resolution resolution of a modern of a modern high resolution transmission electron high resolution transmission electron microscope is closer to microscope is closer to 0.1 nm0.1 nm. .

The reason we are not able to achieve the The reason we are not able to achieve the nearly 100-fold better resolution of 0.003 nearly 100-fold better resolution of 0.003 nm is due to the nm is due to the extremely narrow aperture extremely narrow aperture angles (about 1,000 times smaller than that angles (about 1,000 times smaller than that of the light microscopeof the light microscope) needed by the ) needed by the electron microscope lenses to overcome a electron microscope lenses to overcome a major resolution limiting phenomenon major resolution limiting phenomenon called called spherical aberration. spherical aberration.

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In addition, the In addition, the diffraction phenomenondiffraction phenomenon as well as chromatic aberration and astias well as chromatic aberration and astigmatismgmatism (to be discussed later) all degra (to be discussed later) all degrade the resolution capabilities of the TEM.de the resolution capabilities of the TEM. To appreciate these problems, it is neceTo appreciate these problems, it is necessary to understand ssary to understand how lenses functionhow lenses function..

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Design of Electromagnetic Design of Electromagnetic LensesLenses

Since Since electrons electrons are particles with such are particles with such small masssmall mass that they will be stopped even by that they will be stopped even by gas molecules gas molecules present in the airpresent in the air, glass lenses are of no value in , glass lenses are of no value in an electron microscope. an electron microscope.

However, However, since since electrons have a chargeelectrons have a charge, they can , they can be affected by be affected by magnetic fieldsmagnetic fields..

For example, an electron accelerated through a For example, an electron accelerated through a vacuum will follow a vacuum will follow a helical path when it passes helical path when it passes through a magnetic fieldthrough a magnetic field generated by a generated by a coil of coil of wire with a direct current (DC) running through itwire with a direct current (DC) running through it (see Figure 6.11). (see Figure 6.11).

Such simple electromagnetic coils are termed Such simple electromagnetic coils are termed solenoids.solenoids.

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Figure 6.11Single electron passing throughelectromagnetic lens. Instead of traveling in a straightline along the axis of the lens, the electron is forced bythe magnetic field to follow a helical trajectory that willconverge at a defined focal point after it emerges fromthe lens. Therefore, electromagnets, which are DCpowered, behave similar to converging glass lenses.

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Suppose one illuminates a specimen (the arrow Suppose one illuminates a specimen (the arrow shown in Figure 6.12) with a beam of electrons such shown in Figure 6.12) with a beam of electrons such that some of the that some of the electrons that interact with a electrons that interact with a specimen pointspecimen point ( (A inA in Figure 6.12) are transmitted Figure 6.12) are transmitted through the specimen and enter the through the specimen and enter the electromagnetic lens. electromagnetic lens.

Depending on their precise trajectories as they Depending on their precise trajectories as they enter the magnetic field, they will assume various enter the magnetic field, they will assume various helical pathshelical paths as they speed through the lens. as they speed through the lens.

After leaving the lens, the electrons will focus at After leaving the lens, the electrons will focus at point A'point A' to generate an image point of the to generate an image point of the specimen. specimen.

The distance from the center of the lens to where The distance from the center of the lens to where the electrons converge at the electrons converge at A' represents the focal A' represents the focal lengthlength of the electromagnetic lens of the electromagnetic lens

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Figure 6.12Figure 6.12A group of electrons originating from point A in the specimen A group of electrons originating from point A in the specimen plane pass through an electromagnetic lens to all be focused at plane pass through an electromagnetic lens to all be focused at an appropriate point (A') in the image plane. an appropriate point (A') in the image plane.

The specimen is represented as the heavy arrow in this drawing. The specimen is represented as the heavy arrow in this drawing. The electromagnetic lens behaves as a thin biconvex glass lens asThe electromagnetic lens behaves as a thin biconvex glass lens as

shown in Figure 6.9.shown in Figure 6.9.

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It is possible to change the focal length of an It is possible to change the focal length of an electromagnetic lens by changing the electromagnetic lens by changing the amount of DC current running through the amount of DC current running through the coil of wirecoil of wire. This relationship is expressed in . This relationship is expressed in Equation 6.4: Equation 6.4:

Equation 6.4: Equation 6.4: Focal LengthFocal Length of Electromagnetic Lens of Electromagnetic Lens

f = K (V/if = K (V/i22)) where K = constant based on number of where K = constant based on number of

turns in lens coil wire and geometry of lensturns in lens coil wire and geometry of lens V = accelerating V = accelerating voltagevoltage i = milliamps of i = milliamps of currentcurrent put through coil put through coil

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As the As the accelerating voltageaccelerating voltage of the electron of the electron is is increasedincreased, the , the focal lengthfocal length is also is also increasedincreased since the electrons pass much since the electrons pass much more rapidly through the lens and assume more rapidly through the lens and assume looser helical routes. looser helical routes.

An increase in An increase in current current put through the lens put through the lens coil, however, results in a coil, however, results in a shorter focal shorter focal lengthlength by forcing the electrons to assume by forcing the electrons to assume tighter helical trajectories. tighter helical trajectories.

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Being able to Being able to change the focal lengthchange the focal length of a lens is of pra of a lens is of practical importance, because this is how one can ctical importance, because this is how one can focus afocus an image formed by a lensn image formed by a lens as well as change the as well as change the magnimagnificationfication. . In the light microscope, where the glass lenses are of In the light microscope, where the glass lenses are of a fixed focal length, focussing is done by physically ma fixed focal length, focussing is done by physically moving the specimen into the proper plane of focus for oving the specimen into the proper plane of focus for each objective lens or vice versa. each objective lens or vice versa. Similarly, magnifications are changed by removing an Similarly, magnifications are changed by removing an objective lens of one fixed focal length and replacing iobjective lens of one fixed focal length and replacing it with another. t with another.

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Obviously, the electromagnetic Obviously, the electromagnetic lenses of the electron microscope are lenses of the electron microscope are advantageous advantageous because they permit because they permit one to one to change focal lengths (e.g., change focal lengths (e.g., change focus and magnificationchange focus and magnification) by ) by varying the varying the current running through current running through the lens coilthe lens coil without having to move without having to move the specimen or physically change the specimen or physically change lenses. lenses.

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The The efficiency of the electromagnetic lensefficiency of the electromagnetic lens can can be greatly improved by be greatly improved by concentrating the concentrating the magnetic field strengthmagnetic field strength close to the path of close to the path of the electrons. the electrons.

This is accomplished by This is accomplished by shrouding the coilshrouding the coil on on top, bottom, and side top, bottom, and side with a soft-iron casingwith a soft-iron casing so that the magnetism will run through the so that the magnetism will run through the shroud (Figure 6.13A). shroud (Figure 6.13A).

The strength of the lens is thereby increased. The strength of the lens is thereby increased. (The term (The term soft ironsoft iron refers not only to the refers not only to the hardness of the metal, but also indicates that hardness of the metal, but also indicates that the iron is magnetized only when the the iron is magnetized only when the electromagnetic field is conducted through it.) electromagnetic field is conducted through it.)

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Figure 6.13ADiagram of electromagnetic lens showingsoft-iron casing (shroud) and soft-iron polepiecethat slips down inside bore of lens.

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The The strength of the lensstrength of the lens can be can be further increasedfurther increased by by concenconcentrating the magnetismtrating the magnetism to an even smaller area inside the le to an even smaller area inside the lens bore by means of a liner termed a ns bore by means of a liner termed a polepiecepolepiece (so named b(so named because it sits in the north and south poles of the magnet). ecause it sits in the north and south poles of the magnet). The cylindrical The cylindrical polepiece polepiece (Figure 6.13A and B) consists of (Figure 6.13A and B) consists of uupper and lower cores of soft iron held apart by a nonmagnepper and lower cores of soft iron held apart by a nonmagnetic brass spacertic brass spacer. The magnetic field is now concentrated be. The magnetic field is now concentrated between the top and bottom (north and south) iron componetween the top and bottom (north and south) iron components of the polepiece. nts of the polepiece. These north and south These north and south corescores of the polepiece are bored mu of the polepiece are bored much smaller than the polepiece ch smaller than the polepiece linerliner and and must be as symmetmust be as symmetrical as is mechanically possiblerical as is mechanically possible in order to achieve high res in order to achieve high resolution. olution. In practice, they are In practice, they are rarely perfect and may possess a numbrarely perfect and may possess a number of defectser of defects that may degrade resolving power. that may degrade resolving power.

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Figure 6.13B (top) Photograph of a polepiece that fits into the electromagnetic lens coil shown on the left. The soft-iron casing of the lens coil is removed to reveal the wire windings around the brass spool. (bottom) In the polepiece the north pole is arbitrarily on top, followed by a nonmagnetic brass spacer that holds north and south poles apart.

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Defects in LensesDefects in Lenses A number of imperfections in lenses may reduce A number of imperfections in lenses may reduce

resolution. resolution. AstigmatismAstigmatism results when a lens field is results when a lens field is not not

symmetrical in strengthsymmetrical in strength, but is , but is stronger in one stronger in one plane (north and south, for exampleplane (north and south, for example) and ) and weaker weaker in another (east and westin another (east and west) so that only ) so that only part of the part of the imageimage will be will be in focus at one timein focus at one time (Figure 6.14). (Figure 6.14).

A A point would not be imaged as suchpoint would not be imaged as such, but would , but would appear appear elliptical in shapeelliptical in shape; a ; a crosscross would be would be imaged with either the imaged with either the vertical or horizontal armvertical or horizontal arm, , but not both, in focus at one timebut not both, in focus at one time. .

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Figure 6.14Astigmatism in a lens. Since the lens field isasymmetrically weaker in the north/south plane, objectsoriented along the north/south axis will focus at a longerdistance. By contrast, due to a stronger east/westlens field, objects oriented east/west will come tofocus at a shorter distance from the lens. The effect isthat only some portions of the image (eithernorth/south or east/west) will be in focus at one time.Obviously, resolution will be degraded since the imagewill be focused in only one plane.

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Defects in LensesDefects in Lenses Some Some causes of astigmatismcauses of astigmatism are an are an imperfectlyimperfectly grounground polepiece bore, nonhomogeneous blending of the pd polepiece bore, nonhomogeneous blending of the polepiece metalsolepiece metals, and , and dirt on parts of the columndirt on parts of the column such such as polepieces, apertures, and specimen holders. as polepieces, apertures, and specimen holders. Because it is Because it is impossible to fabricate and maintainimpossible to fabricate and maintain a le a lens with a ns with a perfectly symmetrical lens fieldperfectly symmetrical lens field, it is necessa, it is necessary to ry to correct astigmatismcorrect astigmatism by applying a by applying a correcting fielcorrecting fieldd of the appropriate strength in the proper direction of the appropriate strength in the proper direction tto counteract the asymmetryo counteract the asymmetry. . Such a device is called a Such a device is called a stigmatorstigmator and can be found i and can be found in the n the condenser, objective, and intermediate lensescondenser, objective, and intermediate lenses of of the electron microscope (see Figure 6.35B). the electron microscope (see Figure 6.35B).

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Figure 6.35(A) Conceptual drawing of electromagnetic stigmatorshowing orientation of eight electromagnets around The lens axis. Strength and direction are controlled By adjusting appropriate combinations of magnetsto generate a symmetrical field. The stigmator is Located under the condenser and the objective lens polepieces.(B) Actual stigmator apparatus taken from an Electron microscope. The large arrow indicates one of the eight electromagnetic iron slugs oriented around the central axis. The entire apparatus fits up into the bore of the objective lens so that the area indicated in the large arrow is positioned just under the specimen. The smaller arrow points out individual electrical contacts through which current flows to energize the electromagnets. The close-up photograph (bottom) shows some of The electromagnets that are positioned near the Specimen (arrow).

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Defects in LensesDefects in LensesAstigmatism in a glass lens could be Astigmatism in a glass lens could be

corrected by corrected by regrinding the regrinding the curvature of the lenscurvature of the lens so that the so that the strength is symmetrical, or by strength is symmetrical, or by imposing another lens field of the imposing another lens field of the appropriate strength over one of the appropriate strength over one of the aberrant fields of the original lens—aberrant fields of the original lens—as is done with correcting as is done with correcting eyeglasses. eyeglasses.

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Defects in LensesDefects in Lenses Chromatic aberrationChromatic aberration results when electromagnetic results when electromagnetic

radiations of radiations of different energies convergedifferent energies converge at different at different focal planes. focal planes.

With a With a glass lensglass lens, , shorter wavelength radiations are shorter wavelength radiations are slowedslowed down and refracted more than are longer down and refracted more than are longer wavelengths of light. wavelengths of light.

Effectively, the Effectively, the shorter, more energetic wavelengths shorter, more energetic wavelengths of light come to a shorter focal pointof light come to a shorter focal point than do the than do the longer wavelengths (Figure 6.15).longer wavelengths (Figure 6.15).

In an In an electromagnetic lenselectromagnetic lens, the , the reverse is truereverse is true: : shorter wavelength, more energetic electrons have a shorter wavelength, more energetic electrons have a longer focal pointlonger focal point than do the longer wavelength than do the longer wavelength electrons.electrons.

In both cases, however, In both cases, however, chromatic aberration results chromatic aberration results in the in the enlargement of the focal pointenlargement of the focal point (similar to the (similar to the Airy disc phenomenon caused by diffraction effects) Airy disc phenomenon caused by diffraction effects) with a consequential loss of resolution. with a consequential loss of resolution.

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Figure 6.15Chromatic aberration in a glass lens. Differentwavelengths do not come to focus at the same point.Note how the violet part of the spectrum (gray area)focuses at a shorter distance than does the red part of thespectrum. This results in an enlarged, unsharp pointrather than a smaller, focused one. Resolution of thepoint will be degraded.

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Defects in LensesDefects in Lenses Chromatic aberrationChromatic aberration can be corrected by using a can be corrected by using a

monochromatic sourcemonochromatic source of electromagnetic radiation. of electromagnetic radiation. With glass lenses, one would use a monochromatic With glass lenses, one would use a monochromatic

light (possibly by using a shorter wavelength blue light (possibly by using a shorter wavelength blue filter). filter).

In an electromagnetic lens, one would insure that the In an electromagnetic lens, one would insure that the electrons were of the same energy level by electrons were of the same energy level by carefully stabilizing the accelerating voltagecarefully stabilizing the accelerating voltage and and having having a good vacuum to minimize the energy lossa good vacuum to minimize the energy loss of of the electronsthe electrons as they passed through the column. as they passed through the column.

Thicker specimensThicker specimens give rise to a spectrum of electrons give rise to a spectrum of electrons with with varied energy levelsvaried energy levels and consequently and consequently worsen worsen chromatic aberrationchromatic aberration (Figure 6.16A). (Figure 6.16A).

Thin specimensThin specimens are therefore essential for high are therefore essential for high resolution studies. resolution studies.

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Figure 6.16AChromatic aberration in an overly thick sectionis evidenced by an image that is blurred overall dueto degraded resolution. Figure 6.16BChromatic change of magnification occurs whenan overly thick specimen is viewed at low magnificationswith a low accelerating voltage. Only the central part ofthe image is sharp since the effect is maximal atthe periphery.

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Defects in LensesDefects in Lenses Chromatic change in magnificationChromatic change in magnification occurs when occurs when

thick specimens are viewed at low magnifications thick specimens are viewed at low magnifications using a low accelerating voltage. using a low accelerating voltage.

The The image appears to be sharp in the center, but image appears to be sharp in the center, but becomes progressively out of focus as one moves becomes progressively out of focus as one moves toward the peripherytoward the periphery (Figure 6.16B). (Figure 6.16B).

This is because the lower energy electrons are This is because the lower energy electrons are imaged imaged at a different plane than the higher energy electronsat a different plane than the higher energy electrons. .

The effect is maximal at the periphery of the image, The effect is maximal at the periphery of the image, since these electrons are closer to the lens coils and, since these electrons are closer to the lens coils and, thus, are more affected by the magnetic field. thus, are more affected by the magnetic field.

This problem may be This problem may be minimized by using thinner minimized by using thinner specimens, higher accelerating voltages, higher specimens, higher accelerating voltages, higher magnificationsmagnifications, and , and by correcting any other distortionsby correcting any other distortions that may be present in the lens. that may be present in the lens.

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Defects in LensesDefects in Lenses Spherical aberrationSpherical aberration is due to the is due to the geometry of both ggeometry of both glass and electromagnetic lenseslass and electromagnetic lenses such that rays passin such that rays passing through the g through the periphery of the lens are periphery of the lens are refracted morerefracted more than rays passing along the axisthan rays passing along the axis. . Unfortunately, the various rays do not come to a comUnfortunately, the various rays do not come to a common focal point, resulting in an enlarged, mon focal point, resulting in an enlarged, unsharp poiunsharp pointnt (Figure 6.17A). (Figure 6.17A). At some distance, however, one should At some distance, however, one should encounter the encounter the sharpest possible pointsharpest possible point that would constitute the that would constitute the circlcircle of minimum confusione of minimum confusion (i.e., the smallest Airy disc) a (i.e., the smallest Airy disc) and the practical focal point of the lens. nd the practical focal point of the lens.

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Figure 6.17(A) Spherical aberration in a lens. Peripheral rays are refracted more than central rays, so that all rays do not converge to a common, small focal point. Instead, an enlarged, diffuse spot like the Airy disc will be generated. The vertical line indicates the one point where the point will be smallest (i.e., having the smallest circle of confusion). (B) Correction of spherical aberration with an aperture (here shown inside the lens)to cut out peripheral rays and thereby permit remaining rays to focus at a common small imaging point.

Resolution will be improved since individual Image points in the specimen will be smaller.

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Defects in LensesDefects in Lenses Spherical aberration may be reduced by using an aperSpherical aberration may be reduced by using an aperture to ture to eliminate some of the peripheral rayseliminate some of the peripheral rays (Figure 6. (Figure 6.17B). 17B). Although apertures must be used in the electron micrAlthough apertures must be used in the electron microscope to reduce spherical aberration as much as pososcope to reduce spherical aberration as much as possible, they decrease the aperture sible, they decrease the aperture angle and thereby prangle and thereby prevent the electron microscope from achieving the ultievent the electron microscope from achieving the ultimate resolving power specified in the equation for resmate resolving power specified in the equation for resolutionolution (Equation 6.1). r= 0.612 λ/n (sin α) (Equation 6.1). r= 0.612 λ/n (sin α) In addition, the worsening of resolution as a result of In addition, the worsening of resolution as a result of using a longer focal length lens is shown in Equation 6.using a longer focal length lens is shown in Equation 6.5. 5.

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Determine resolving powerDetermine resolving powerRadius of Airy Disc: the Radius of Airy Disc: the radius of the Airy radius of the Airy discdisc as measured to the first dark ring is as measured to the first dark ring is express by Equation 6.1:express by Equation 6.1:

r= 0.612 λ/r= 0.612 λ/nn (sin α) (sin α)

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Defects in LensesDefects in LensesEquation 6.5: Equation 6.5: Limit of ResolutionLimit of Resolution, ds, I, ds, Imposed by mposed by Spherical AberrationSpherical Aberration

DDss = k = kss f f αα0033where k = a constant related to lens charwhere k = a constant related to lens characteristicsacteristics f = focal length of lensf = focal length of lensα = aperture angle of lens, normally the α = aperture angle of lens, normally the objective lensobjective lens

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Defects in LensesDefects in Lenses From Equation 6.5, we see why a From Equation 6.5, we see why a short focal short focal

length lens combined with a smaller length lens combined with a smaller apertureaperture (to generate a smaller aperture (to generate a smaller aperture angle) will help to angle) will help to reduce the degradation of reduce the degradation of resolutionresolution caused by spherical aberration. caused by spherical aberration.

Consequently, Consequently, smaller aperturessmaller apertures are are generally more desirable than generally more desirable than larger oneslarger ones to to improve improve resolution—in spite of the resolution—in spite of the theoretical advantage offered by large theoretical advantage offered by large apertures as indicated in Equation 6.1. apertures as indicated in Equation 6.1.

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Defects in LensesDefects in Lenses Certain types of image Certain types of image distortionsdistortions may arise may arise

when spherical aberration occurs in the final when spherical aberration occurs in the final imaging (or projector) lenses of the imaging (or projector) lenses of the transmission electron microscope. transmission electron microscope.

Since peripheral electrons are refracted to a Since peripheral electrons are refracted to a greater extent than central rays, the image greater extent than central rays, the image formed by these formed by these peripheral electrons will be peripheral electrons will be at a greater magnificationat a greater magnification and in and in a different a different focal planefocal plane than the image generated from than the image generated from more centrally positioned electrons. more centrally positioned electrons.

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Defects in LensesDefects in Lenses A A grid of lines would not be imaged as squaregrid of lines would not be imaged as square (Figure (Figure 6.18A), but would assume the shape of a sunken pillo6.18A), but would assume the shape of a sunken pillow, hence the name w, hence the name pincushion distortionpincushion distortion (Figure 6.18 (Figure 6.18B). B). This type of distortion may occur when attempting to This type of distortion may occur when attempting to operate the operate the transmission electron microscopetransmission electron microscope at exce at excessively ssively low magnificationslow magnifications. . Another type of imperfection, Another type of imperfection, barrel distortionbarrel distortion,, occurs occurs when one attempts to use an electromagnetic lens in when one attempts to use an electromagnetic lens in a a demagnifying modedemagnifying mode rather than the normal magnifyi rather than the normal magnifying mode. ng mode. In this case, the central part of the image is magnified In this case, the central part of the image is magnified more than the periphery so that the gridwork assumes more than the periphery so that the gridwork assumes a swollen or barrel shape (Figure 6.18C). a swollen or barrel shape (Figure 6.18C).

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Figure 6.18 Distortions in a lens.

(A) Normal image of grid pattern.

(B) Image with pincushion distortion.(C) Image with barrel distortion.

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Defects in LensesDefects in Lenses Fortunately, it isFortunately, it is possible possible to neutralize one type of dist to neutralize one type of distortion with the other. ortion with the other. For instance, if one For instance, if one projector lensprojector lens is displaying is displaying excessivexcessive pincushion distortione pincushion distortion, it is possible to operate anothe, it is possible to operate another projector lens in the demagnifying mode to introduce r projector lens in the demagnifying mode to introduce an opposing barrel distortion. an opposing barrel distortion. The lens systems of modern electron microscopes are The lens systems of modern electron microscopes are designed to automatically counterbalancedesigned to automatically counterbalance the various t the various types of distortions throughout a wide magnification raypes of distortions throughout a wide magnification range. nge. In older microscopes, however, one must take care not In older microscopes, however, one must take care not to introduce these distortions in the lower magnificatioto introduce these distortions in the lower magnification range. n range.

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Defects in LensesDefects in Lenses A curious phenomenon called A curious phenomenon called image rotationimage rotation will be noticed will be noticed

as one changes magnification in older microscopes. as one changes magnification in older microscopes. This occurs because the electrons follow a This occurs because the electrons follow a spiral pathspiral path

through the lenses, and the spiral shifts as the strength of through the lenses, and the spiral shifts as the strength of the lens is varied. the lens is varied.

Image rotation not only results in rotation of the image on Image rotation not only results in rotation of the image on the viewing screen as one increases magnification, but also the viewing screen as one increases magnification, but also exaggerates the effects of distortionexaggerates the effects of distortion. .

It is possible to It is possible to minimize or eliminate image rotation minimize or eliminate image rotation entirelyentirely by ensuring that a by ensuring that a series of lensesseries of lenses have have opposing opposing rotations rather than all having rotations in the same rotations rather than all having rotations in the same directiondirection. .

This is accomplished by This is accomplished by running the lens current through running the lens current through the coilthe coil in the opposite direction (i.e., reversing polarity) in the opposite direction (i.e., reversing polarity) and is a principle utilized in some of the newer transmission and is a principle utilized in some of the newer transmission electron microscopes.electron microscopes.

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MagnificationMagnification Besides forming images with high resolution, Besides forming images with high resolution,

the the lenses lenses of the electron microscope are able of the electron microscope are able to further to further magnify these imagesmagnify these images. .

Magnification refers to the Magnification refers to the degree of degree of enlargementenlargement of the diameter of a final image of the diameter of a final image compared to the original. compared to the original.

In practice, magnification equals In practice, magnification equals a distance a distance measured between two pointsmeasured between two points on an image on an image divided by the distance measured between divided by the distance measured between these same two points on the original object, these same two points on the original object, or or

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MagnificationMagnificationConsequently, if the Consequently, if the image distance betimage distance between two pointsween two points measures 25.5 mm whil measures 25.5 mm while the e the distance between these same two distance between these same two points on the objectpoints on the object measures 5 mm, th measures 5 mm, then the magnification is en the magnification is

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MagnificationMagnificationAs will be discussed later, there are at As will be discussed later, there are at

least three magnifying lenses in an least three magnifying lenses in an electron microscope: the electron microscope: the objective, objective, intermediate,intermediate, and and projector lensesprojector lenses..

The final magnification is calculated as The final magnification is calculated as the product of the individual magnifying the product of the individual magnifying powers of all of the lenses in the powers of all of the lenses in the system as shown in Equation 6.6. system as shown in Equation 6.6.

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MagnificationMagnificationEquation 6.6: Calculation of Total MagnEquation 6.6: Calculation of Total Magnification, Mification, MTT, of the TEM, of the TEM

where: Mwhere: MTT = total magnification or mag = total magnification or magMMOO = mag of = mag of objectiveobjective lens lensMMII = mag of = mag of intermediateintermediate lens lensMMPP = mag of = mag of projector projector lens(es)lens(es)

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MagnificationMagnificationFor example, if the transmission For example, if the transmission

electron microscope is operating in electron microscope is operating in the high magnification mode, typical the high magnification mode, typical values for the respective lenses might values for the respective lenses might be: 200 × 50 × 20 = 200,000×. be: 200 × 50 × 20 = 200,000×.

If one were to operate the microscope If one were to operate the microscope in the low magnification mode, in the low magnification mode, perhaps the values would be: 50 × 0.5 perhaps the values would be: 50 × 0.5 × 50 = 1.250×. × 50 = 1.250×.

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MagnificationMagnification Intermediate magnifications may be produced by Intermediate magnifications may be produced by

varying the current to the various lenses. varying the current to the various lenses. Sometimes, it is desirable to view as much of the Sometimes, it is desirable to view as much of the

specimen as possible in order to evaluate quickly specimen as possible in order to evaluate quickly the quality of the preparation or to locate a the quality of the preparation or to locate a particular portion of the specimen. particular portion of the specimen.

In this case, an extremely low magnification is In this case, an extremely low magnification is obtained by placing the microscope in the obtained by placing the microscope in the scan scan magnificationmagnification mode, which can be accomplished by mode, which can be accomplished by shutting off the objective lens and using the next shutting off the objective lens and using the next lens (the intermediate lens) as the imaging lens as lens (the intermediate lens) as the imaging lens as follows: 1 × 0.5 × 100 = 50×. follows: 1 × 0.5 × 100 = 50×.

Of all the lenses used to change magnification, the Of all the lenses used to change magnification, the objective lensobjective lens is used the is used the leastleast. .

Normally it is maintained at around 50 or 100× Normally it is maintained at around 50 or 100× while the other lenses are varied. while the other lenses are varied.

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MagnificationMagnification Although it is theoretically possible to Although it is theoretically possible to

increase the magnification indefinitely, the increase the magnification indefinitely, the quality of the image magnified is quality of the image magnified is dependent on the dependent on the resolving power of the resolving power of the lenses in the systemlenses in the system. .

Consequently, the term Consequently, the term useful useful magnificationmagnification is used to define the is used to define the maximum magnification that should be maximum magnification that should be used for a particular optical system. It is used for a particular optical system. It is defined by the formula in Equation 6.7. defined by the formula in Equation 6.7.

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MagnificationMagnificationEquation 6.7: Useful MagnificationEquation 6.7: Useful Magnification

185185

MagnificationMagnification In the case of the light microscope, a typical value woIn the case of the light microscope, a typical value would be uld be 1,000×1,000× because the resolving power of the hu because the resolving power of the human eye is about man eye is about 0.2 mm0.2 mm, while the resolving power of , while the resolving power of the light microscope is approximately the light microscope is approximately 0.2 0.2 μμmm. . An electron microscope with a resolving power of An electron microscope with a resolving power of 0.2 0.2 nmnm could be expected to have a top magnification of a could be expected to have a top magnification of approximately pproximately 1,000,000×,1,000,000×, or a thousand times greate or a thousand times greater than the light microscope. r than the light microscope. In practice, due to the In practice, due to the diminished illumination at such diminished illumination at such high magnificationshigh magnifications, microscopists would probably ta, microscopists would probably take the micrograph at a magnification of ke the micrograph at a magnification of 250,000× and 250,000× and photographically enlarge the negative to the needed photographically enlarge the negative to the needed magnificationmagnification. . However, only rarely do biologists need such high maHowever, only rarely do biologists need such high magnifications. gnifications.

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MagnificationMagnification Depending on the model of TEM, the Depending on the model of TEM, the

magnification changes may occur as a series of magnification changes may occur as a series of discrete steps or as an infinitely variable or discrete steps or as an infinitely variable or ''zoom" magnification series.''zoom" magnification series.

Modern electron microscopes have Modern electron microscopes have digital digital displaysdisplays that give the approximate total that give the approximate total magnification when one varies the magnification magnification when one varies the magnification control. control.

Older microscopes usually have an analog gauge Older microscopes usually have an analog gauge that may either read the current to one lens, or that may either read the current to one lens, or they may have a magnification knob with a series they may have a magnification knob with a series of click stops that may be correlated to a of click stops that may be correlated to a particular magnification. particular magnification.

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MagnificationMagnification All microscopes (including light microscopes) All microscopes (including light microscopes)

must be calibrated in order to determine must be calibrated in order to determine more accurately the total magnification, more accurately the total magnification, since a since a number of variablesnumber of variables may cause the may cause the magnification to vary by magnification to vary by as much as 20 to as much as 20 to 30%30% over a short period of time. over a short period of time.

Even modern instruments with direct Even modern instruments with direct reading digital displays are guaranteed to be reading digital displays are guaranteed to be accurate to only accurate to only ± 5 to 10%± 5 to 10% of the stated of the stated values. values.

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Design of the Transmission Design of the Transmission Electron MicroscopeElectron Microscope

Comparison of Light Microscope Comparison of Light Microscope to Transmission Electron to Transmission Electron MicroscopeMicroscope

Basic Systems Making Up a Basic Systems Making Up a Transmission Electron Transmission Electron MicroscopeMicroscope

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Comparison of Light Microscope Comparison of Light Microscope to Transmission Electron to Transmission Electron

MicroscopeMicroscopeThe transmission electron microscope The transmission electron microscope

is similar in many ways to the is similar in many ways to the compound light microscope. compound light microscope.

For instance, in both microscopes, For instance, in both microscopes, electromagnetic radiations originating electromagnetic radiations originating from a tungsten filament are from a tungsten filament are converged ontoconverged onto a thin specimen by a thin specimen by means of a means of a condenser lens systemcondenser lens system. .

190190


MicroscopeMicroscope The illumination transmitted through the The illumination transmitted through the

specimen is focused into an specimen is focused into an image and image and magnified firstmagnified first by an by an objective lensobjective lens and and then further magnified by a series of then further magnified by a series of intermediate and projector lensesintermediate and projector lenses until the until the final image is viewed (Figure 6.19). final image is viewed (Figure 6.19).

Both kinds of microscope may Both kinds of microscope may record record images using a silver-based photographic images using a silver-based photographic emulsionemulsion since it is sensitive to both types since it is sensitive to both types of radiations of radiations

191191

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MicroscopeMicroscope Of course, the lenses of light microscopes Of course, the lenses of light microscopes

are composed of glass or quartz rather are composed of glass or quartz rather than the electromagnetic solenoids used in than the electromagnetic solenoids used in electron microscopes. electron microscopes.

Electron microscopes require an Electron microscopes require an elaborate elaborate vacuum systemvacuum system to to remove interfering air remove interfering air moleculesmolecules that would impede the flow of that would impede the flow of electrons down the column. electrons down the column.

Such Such high vacuumshigh vacuums are necessary from the are necessary from the point of origin of the electrons (the point of origin of the electrons (the filament) up to, and usually including, the filament) up to, and usually including, the photographic film. photographic film.

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MicroscopeMicroscopeSince the specimen is also subjected to tSince the specimen is also subjected to these high vacuums, hese high vacuums, living specimens woliving specimens would be rapidly dehydrateduld be rapidly dehydrated if placed direc if placed directly into the electron microscope. tly into the electron microscope. Nonetheless, it is Nonetheless, it is possible possible to view to view rapidlrapidly frozen, hydrated, thin specimensy frozen, hydrated, thin specimens by us by using ing cryostagescryostages that maintain the that maintain the frozen sfrozen state of cellular watertate of cellular water even under bomba even under bombardment by the electron beam. rdment by the electron beam.

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Thank you!Thank you!

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Checking PerformanceChecking Performance Alignment Alignment It is possible to check and make minor corrections to tIt is possible to check and make minor corrections to the alignment of the electron microscope in less than 1he alignment of the electron microscope in less than 10 minutes, as follows: 0 minutes, as follows: Without a specimen in the TEM, verify that the filamenWithout a specimen in the TEM, verify that the filament cloud is symmetrical, the condenser lens is stigmatet cloud is symmetrical, the condenser lens is stigmated, and the C1 aperture is centered. Check that the C2 ad, and the C1 aperture is centered. Check that the C2 aperture is centered by varying the current to the C2 leperture is centered by varying the current to the C2 lens and observing that the illumination stays centered. ns and observing that the illumination stays centered. Insert a holey film and set the microscope at a low maInsert a holey film and set the microscope at a low magnification. Bring the illumination to the smallest possgnification. Bring the illumination to the smallest possible crossover spot size and then increase to the top mible crossover spot size and then increase to the top magnification. Both the illumination spot and the image agnification. Both the illumination spot and the image should stay close to the center of the screen. should stay close to the center of the screen.

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Checking PerformanceChecking Performance If either move off screen, then adjustments will be neeIf either move off screen, then adjustments will be needed. Now, repeat this process using the largest spot seded. Now, repeat this process using the largest spot selectable by C1 and make corrections if the illuminatiolectable by C1 and make corrections if the illumination does not stay centered. Insert a specimen and tilt thn does not stay centered. Insert a specimen and tilt the stage ±10° to see that the specimen stays centered. e stage ±10° to see that the specimen stays centered. Wobble the high voltage at a high magnification and oWobble the high voltage at a high magnification and observe that the focused image rotates around the centbserve that the focused image rotates around the center of the screen. Go to the diffraction mode with the iner of the screen. Go to the diffraction mode with the intermediate lens and observe that the caustic pattern itermediate lens and observe that the caustic pattern is centered. Check centration of the objective aperture s centered. Check centration of the objective aperture while in the diffraction mode. Finally, stigmate the miwhile in the diffraction mode. Finally, stigmate the microscope if necessary.croscope if necessary.

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Electrical StabilityElectrical Stability The microscopist should be aware that a The microscopist should be aware that a recently turned on microscope will be sorecently turned on microscope will be somewhat unstable until the high voltage mewhat unstable until the high voltage and lens circuits have warmed up for perand lens circuits have warmed up for perhaps 30 to 60 minutes. If imaging problehaps 30 to 60 minutes. If imaging problems are still encountered and instabilities ms are still encountered and instabilities in the microscope are suspected, several in the microscope are suspected, several areas may be checked as follows: areas may be checked as follows:

200200

Electrical StabilityElectrical Stability Problems with the filament may be checked bProblems with the filament may be checked by undersaturating the filament and carefully oy undersaturating the filament and carefully observing its focused image on the screen. Chabserving its focused image on the screen. Changes in the size or brightness of the halo may inges in the size or brightness of the halo may indicate problems with the vacuum, bias contrndicate problems with the vacuum, bias controls, contamination in the gun area, or it may inols, contamination in the gun area, or it may indicate that a weak filament is about to burn oudicate that a weak filament is about to burn out. Instabilities in the high voltage, diffraction, ot. Instabilities in the high voltage, diffraction, or projector lenses may be detected by observir projector lenses may be detected by observing the caustic image. If the caustic image channg the caustic image. If the caustic image changes in sharpness or moves, then instabilities in ges in sharpness or moves, then instabilities in these circuits should be pursued further. these circuits should be pursued further.

201201

Electrical StabilityElectrical Stability Objective lens instabilities may be detected by observiObjective lens instabilities may be detected by observing an overfocused Fresnel fringe in a holey film. Shoulng an overfocused Fresnel fringe in a holey film. Should the fringe change in any way, objective lens circuits d the fringe change in any way, objective lens circuits should be checked. All electron microscopes have builshould be checked. All electron microscopes have built-in gauges or test points in circuit boards that may be t-in gauges or test points in circuit boards that may be monitored using appropriate testing equipment. Howmonitored using appropriate testing equipment. However, unless one has a thorough understanding of the ever, unless one has a thorough understanding of the process and an appreciation of the dangers involved, tprocess and an appreciation of the dangers involved, this is best left to trained personnel. At least one will hhis is best left to trained personnel. At least one will have diagnosed the problem as a microscope—not a spave diagnosed the problem as a microscope—not a specimen—problem. ecimen—problem.

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Image DriftImage Drift Gradual shifting of the image, usually Gradual shifting of the image, usually

in one direction, is a common and in one direction, is a common and annoying problem, encountered annoying problem, encountered especially when support films are not especially when support films are not used. used.

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Image DriftImage Drift The most common cause of image drift is a dirty The most common cause of image drift is a dirty

specimen holder. specimen holder. All holders should be checked and cleaned on a All holders should be checked and cleaned on a

routine basis. Drift might also be caused by heating routine basis. Drift might also be caused by heating of the specimen due to excessive beam irradiation, of the specimen due to excessive beam irradiation, in which case a smaller spot size, condenser in which case a smaller spot size, condenser aperture, or less filament emission may be tried. aperture, or less filament emission may be tried.

If the section or plastic substrate is not firmly If the section or plastic substrate is not firmly attached to the grid, it will move as the grid heats attached to the grid, it will move as the grid heats up—the amount of movement is related to the up—the amount of movement is related to the beam intensity and the area illuminated. beam intensity and the area illuminated.

This may be confirmed by examining a test This may be confirmed by examining a test specimen known to be thermally stable. specimen known to be thermally stable. Contamination in the area above the specimen may Contamination in the area above the specimen may lead to charging and shifting of the image as the lead to charging and shifting of the image as the static is discharged. static is discharged.

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Image DriftImage Drift This should be suspected if the image shifts This should be suspected if the image shifts

as the C2 illumination level is changed. as the C2 illumination level is changed. Moderate drift of the specimen on the screen Moderate drift of the specimen on the screen may be seen through the viewing binoculars. may be seen through the viewing binoculars.

An easy way to test a specimen for drift is to An easy way to test a specimen for drift is to place a recognizable specimen structure place a recognizable specimen structure next to a fixed point on the viewing screen next to a fixed point on the viewing screen and observe the movement of the specimen and observe the movement of the specimen over an interval 2 to 3 times greater than over an interval 2 to 3 times greater than the exposure time for the negative. the exposure time for the negative.

205205

Image DriftImage Drift Drift may be documented by taking an exposure of an Drift may be documented by taking an exposure of an overfocused hole, waiting for 1 to 2 minutes with the soverfocused hole, waiting for 1 to 2 minutes with the specimen still being irradiated, and then taking a seconpecimen still being irradiated, and then taking a second exposure on the film. This double-exposed film is thd exposure on the film. This double-exposed film is then developed and the distance traveled by the hole men developed and the distance traveled by the hole may be converted into nanometers traveled per second ay be converted into nanometers traveled per second (Figure 6.53). For example, if one is hoping to resolve 1 (Figure 6.53). For example, if one is hoping to resolve 1 nm and the exposure time is 4 seconds, then the specinm and the exposure time is 4 seconds, then the specimen should not drift any more than 1 nm per 4 secondmen should not drift any more than 1 nm per 4 seconds. At a magnification of 500,000×, the drift should not s. At a magnification of 500,000×, the drift should not exceed 5 mm over 4 seconds. (See the section "Magniexceed 5 mm over 4 seconds. (See the section "Magnification Calibration" later in this chapter.) fication Calibration" later in this chapter.)

206206

Figure 6.53Drift in an electron microscope. Two exposures aremade on the same negative with an interval of 1 to 2minutes between exposures. The rate of drift may becalculated based on the distance moved over theintervening time period.

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ContaminationContaminationDeposition of contaminants onto the spDeposition of contaminants onto the specimen that is being subjected to electroecimen that is being subjected to electron bombardment will degrade resolution. n bombardment will degrade resolution. One should be prepared to quantitate thOne should be prepared to quantitate the rate of contamination in order to detere rate of contamination in order to determine when it has become unacceptable mine when it has become unacceptable for the resolution level needed. for the resolution level needed.

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ContaminationContamination The contamination rate may be quantitated by doublThe contamination rate may be quantitated by double-exposing a hole in a test specimen at a magnificatioe-exposing a hole in a test specimen at a magnification of 250,000× as described in the previous paragraph. n of 250,000× as described in the previous paragraph. The decrease in the diameter of the hole, by depositioThe decrease in the diameter of the hole, by deposition of contamination along the rim, is an excellent indicn of contamination along the rim, is an excellent indicator of the rate of contamination. A 2 to 3 minute expoator of the rate of contamination. A 2 to 3 minute exposure should indicate a contamination rate of less than sure should indicate a contamination rate of less than 0.01 nm per second with all anticontaminators in oper0.01 nm per second with all anticontaminators in operation. Without a specimen anticontaminator, the rates ation. Without a specimen anticontaminator, the rates may be ten times higher even in a clean microscope. may be ten times higher even in a clean microscope.

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ContaminationContamination It should be realized that a recently cleaned miIt should be realized that a recently cleaned microscope column is actually quite contaminatcroscope column is actually quite contaminated with organic vapors used in the cleaning pred with organic vapors used in the cleaning procedure, so that the microscope should be allocedure, so that the microscope should be allowed to remain in high vacuum for one full daowed to remain in high vacuum for one full day in order to remove these molecules. The antiy in order to remove these molecules. The anticontaminators on the diffusion pumps should contaminators on the diffusion pumps should be filled with liquid nitrogen, but the specimebe filled with liquid nitrogen, but the specimen anticontaminator should not be chilled durin anticontaminator should not be chilled during that time, since it will quickly load up with cng that time, since it will quickly load up with contaminants that may later be released onto tontaminants that may later be released onto the specimen. he specimen.

210210

MagnificationMagnification It is necessary to calibrate the It is necessary to calibrate the

magnification settings of all electron magnification settings of all electron microscopes, since different microscopes, since different mechanical and electronic alterations mechanical and electronic alterations will result in significant variations. will result in significant variations. Even in the most modern of Even in the most modern of microscopes, some manufacturers microscopes, some manufacturers warrant the figure displayed to be warrant the figure displayed to be within only ± 10% of the actual value.within only ± 10% of the actual value.

211211

MagnificationMagnification Although it is possible to calibrate the entire Although it is possible to calibrate the entire

magnification range in the microscope, this may not magnification range in the microscope, this may not be necessary if one uses only certain settings. The be necessary if one uses only certain settings. The frequency of calibration varies with the individual frequency of calibration varies with the individual need for accuracy, as well as with the servicing need for accuracy, as well as with the servicing intervals for major systems in the microscope. Once intervals for major systems in the microscope. Once or twice a year may be sufficient for routine work or twice a year may be sufficient for routine work where high accuracy is not required. In critical where high accuracy is not required. In critical operations, calibration should take place during each operations, calibration should take place during each use of the microscope or, alternately, one may use of the microscope or, alternately, one may include a standard of known size on the same grid include a standard of known size on the same grid as the specimen. A number of situations may cause as the specimen. A number of situations may cause a change in previously determined magnification a change in previously determined magnification figures. figures.

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MagnificationMagnificationAfter After cleaningcleaning operations involving the p operations involving the polepieces, specimen stage, or specimen olepieces, specimen stage, or specimen holders, magnifications should be recaliholders, magnifications should be recalibrated. brated. ServicingServicing of electronics may affect magn of electronics may affect magnifications if lens current or reference circifications if lens current or reference circuits were repaired. uits were repaired.

213213

MagnificationMagnification A major cause of erroneous figures arises from lens A major cause of erroneous figures arises from lens hyhysteresissteresis (also called (also called remanence),remanence), or the residual magn or the residual magnetism left in the soft-iron polepieces even after the eleetism left in the soft-iron polepieces even after the electromagnetic field strength has been changed. This mctromagnetic field strength has been changed. This may be minimized by starting the magnification calibratay be minimized by starting the magnification calibration series at a low magnification and taking micrograpion series at a low magnification and taking micrographs at increasingly higher magnifications. Similarly, the hs at increasingly higher magnifications. Similarly, the low- to high-magnification scheme should be followelow- to high-magnification scheme should be followed when viewing and recording images. One should alsd when viewing and recording images. One should also verify that the calibration is accurate at different acco verify that the calibration is accurate at different accelerating voltages. elerating voltages.

214214

MagnificationMagnification The placement of the The placement of the specimen heightspecimen height in the objective in the objective lens is very critical. Since the position of the grid must lens is very critical. Since the position of the grid must be within 40 mm for accurate replication of a particulabe within 40 mm for accurate replication of a particular magnification setting, the grid must be perfectly flat r magnification setting, the grid must be perfectly flat and the z-axis (specimen height) must be accurately sand the z-axis (specimen height) must be accurately set. This is a significant consideration with a side-entry et. This is a significant consideration with a side-entry stage since the z-axis is adjustable over a very wide rastage since the z-axis is adjustable over a very wide range. If the stage is tilted, the x and y traversement will nge. If the stage is tilted, the x and y traversement will result in a change of specimen height. Bent grids shouresult in a change of specimen height. Bent grids should be avoided, and the specimen should always be on ld be avoided, and the specimen should always be on the same side of the grid relative to the beam the same side of the grid relative to the beam

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Magnification CalibrationMagnification Calibration Calibration may be accomplished by first acquiring acCalibration may be accomplished by first acquiring accurate standards with established sizes and propertiecurate standards with established sizes and properties. One convenient method that may be used to calibras. One convenient method that may be used to calibrate the entire magnification range in the TEM involves tte the entire magnification range in the TEM involves the use of a commercially prepared, crosssection of a she use of a commercially prepared, crosssection of a single-crystal, semiconductor wafer. The standard, callingle-crystal, semiconductor wafer. The standard, called MAG*I*CALä, covers magnifications from 1,000× to ed MAG*I*CALä, covers magnifications from 1,000× to 1,000,000×. When viewed in the TEM, the standard ap1,000,000×. When viewed in the TEM, the standard appears as a series of alternating light (silicon) and dark pears as a series of alternating light (silicon) and dark (SiGe alloy) bands of accurately determined thickness(SiGe alloy) bands of accurately determined thicknesses. Since the series of bands range from 10 nm to over es. Since the series of bands range from 10 nm to over 5 mm, calibrations are accurate over the wide range 5 mm, calibrations are accurate over the wide range mentioned. To determine the magnification (M) based mentioned. To determine the magnification (M) based on the standard, one uses the simple equation below: on the standard, one uses the simple equation below:

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Magnification CalibrationMagnification Calibration

For example, on a negative, if one For example, on a negative, if one measured the spacing between the measured the spacing between the bands to be 10 mm, while the bands to be 10 mm, while the distance is known to be 10 nm on the distance is known to be 10 nm on the standard, then the magnification is standard, then the magnification is one million times, or: one million times, or:

217217

Magnification CalibrationMagnification Calibration Using conventional methods of calibration, however, more Using conventional methods of calibration, however, more

than one standard will be needed to cover the entire than one standard will be needed to cover the entire magnification range. For instance, from the lowest magnification range. For instance, from the lowest magnifications up to perhaps 40,000 to 50,000×, one may magnifications up to perhaps 40,000 to 50,000×, one may use commercially available cross-ruled use commercially available cross-ruled diffraction grating diffraction grating replicasreplicas with 2,160 lines/mm (Figure 6.54). The procedure with 2,160 lines/mm (Figure 6.54). The procedure followed is to focus carefully on the granularity of the followed is to focus carefully on the granularity of the grating and take an electron micrograph at the desired grating and take an electron micrograph at the desired settings. After development, the negatives are placed on an settings. After development, the negatives are placed on an illuminated view box and distances measured in illuminated view box and distances measured in millimeters. Measurements should be made from the same millimeters. Measurements should be made from the same relative positions in each line (e.g., middle of the dark band relative positions in each line (e.g., middle of the dark band of one line to a similar position on the second line). One of one line to a similar position on the second line). One then counts the number of spaces included in the specific then counts the number of spaces included in the specific millimeter distance measured. These values are substituted millimeter distance measured. These values are substituted into Equation 6.10. into Equation 6.10.

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Magnification CalibrationMagnification CalibrationEquation 6.10: Magnification Equation 6.10: Magnification

Calculation from Diffraction Calculation from Diffraction Grating ReplicaGrating Replica

where M = magnificationwhere M = magnificationA = distance in mm between lines on A = distance in mm between lines on

electron micrographelectron micrographB = number of spaces between linesB = number of spaces between lines

219219

Magnification CalibrationMagnification Calibration Several Several sources of errorsources of error exist with the diffraction grati exist with the diffraction grating method. The grating is a platinum/carbon replica ong method. The grating is a platinum/carbon replica of a standard optical grating and may have undergone f a standard optical grating and may have undergone some distortion during the mounting on the grid, thersome distortion during the mounting on the grid, thereby affecting the accuracy of measurements. Generalleby affecting the accuracy of measurements. Generally "waffle" gratings are preferred to the parallel line gry "waffle" gratings are preferred to the parallel line gratings, since the crossed lines permit measurements iatings, since the crossed lines permit measurements in both directtions to increase the accuracy. In order to n both directtions to increase the accuracy. In order to have an accuracy of ± 2%, it is necessary to include at have an accuracy of ± 2%, it is necessary to include at least 10 spaces in the distance measured. In practical least 10 spaces in the distance measured. In practical terms, this means that gratings give a ± 2% error only terms, this means that gratings give a ± 2% error only up to a magnification of 25,000×. up to a magnification of 25,000×.

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Magnification CalibrationMagnification Calibration However, if a ± 5% variability is acceptable, then they However, if a ± 5% variability is acceptable, then they may be used to 50,000 to 60,000×. At the higher magnmay be used to 50,000 to 60,000×. At the higher magnifications, in order to minimize the error involved in coifications, in order to minimize the error involved in counting only a fragment of a space one measures a smaunting only a fragment of a space one measures a small piece of debris at a known accurate magnification all piece of debris at a known accurate magnification and then remeasures this same object at the unknown nd then remeasures this same object at the unknown magnification. The amount of enlargement between tmagnification. The amount of enlargement between the two is the factor by which the known setting is multhe two is the factor by which the known setting is multiplied. An object measuring 5 mm at 20,000× and now iplied. An object measuring 5 mm at 20,000× and now measuring 12.5 mm is 2.5× larger, so that the new mameasuring 12.5 mm is 2.5× larger, so that the new magnification is 2.5× 20,000 = 50,000×. gnification is 2.5× 20,000 = 50,000×.

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Magnification CalibrationMagnification Calibration Above 50,000×, one must resort to Above 50,000×, one must resort to organometallic cryorganometallic crystalsstals with established lattice spacings. A good standar with established lattice spacings. A good standard is beef liver catalase prepared by placing a drop of td is beef liver catalase prepared by placing a drop of the catalase suspension on a coated grid for 5 to 10 seche catalase suspension on a coated grid for 5 to 10 seconds and then negatively staining the specimen by floonds and then negatively staining the specimen by floating the grid on a drop of 2% aqueous solution of amating the grid on a drop of 2% aqueous solution of ammonium molybdate or potassium phosphotungstate monium molybdate or potassium phosphotungstate (see Chapter 5). The largest lattice, beef catalase, has (see Chapter 5). The largest lattice, beef catalase, has spacings of 8.8 nm. Alternative standards might includspacings of 8.8 nm. Alternative standards might include bacteriophage or tobacco mosaic virus particles thae bacteriophage or tobacco mosaic virus particles that may be obtained from colleagues or purchased from t may be obtained from colleagues or purchased from the American Type Culture Collection. the American Type Culture Collection.

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ResolutionResolution Most electron microscopes have a guaranteed Most electron microscopes have a guaranteed optimum resolution figure that was verified by optimum resolution figure that was verified by the manufacturer usually upon installation of tthe manufacturer usually upon installation of the microscope. he microscope. As long as one obtains satisfactory micrographAs long as one obtains satisfactory micrographs, it may be mistakenly assumed that the resols, it may be mistakenly assumed that the resolving power has not changed. One should be aving power has not changed. One should be aware of several methods to verify this, since thware of several methods to verify this, since the degradation of resolving power may be so ine degradation of resolving power may be so insidious as to go unnoticed until the quality of sidious as to go unnoticed until the quality of work is brought into question, perhaps to the work is brought into question, perhaps to the embarrassment of the microscopist.embarrassment of the microscopist.

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Figure 6.54Magnification calibration standard. This series of micrographs are of a standard diffraction grating containing2,160 lines/mm. The magnifications were calculated to be 10,400×; 21,000×; and 30,200×, respectively.

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ResolutionResolution The The point-to-point methodpoint-to-point method is the most readily accepte is the most readily accepted method since it graphically demonstrates the ability d method since it graphically demonstrates the ability of the microscope to visualize two fine points separatof the microscope to visualize two fine points separated by a specific distance. As a test specimen, one may ed by a specific distance. As a test specimen, one may use either a thin carbon film alone or with a thin film ouse either a thin carbon film alone or with a thin film of platinum-irridium alloy evaporated onto the carbon. f platinum-irridium alloy evaporated onto the carbon. A series of micrographs are made at the top magnificaA series of micrographs are made at the top magnification needed to demonstrate the resolution (300,000 to tion needed to demonstrate the resolution (300,000 to 500,000×). Even if all conditions are perfect, it is very 500,000×). Even if all conditions are perfect, it is very difficult to obtain satisfactory results from a single midifficult to obtain satisfactory results from a single micrograph, since precise focusing is essential. Consequcrograph, since precise focusing is essential. Consequently, a ently, a through-focus seriesthrough-focus series is made by first focusing t is made by first focusing the image as best as possible and then backing off the he image as best as possible and then backing off the finest focus knob counterclockwise by two click stops. finest focus knob counterclockwise by two click stops.

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ResolutionResolution Micrographs are then rapidly taken, advancing the Micrographs are then rapidly taken, advancing the

focus clockwise for each exposure until a total of five focus clockwise for each exposure until a total of five shots have been made. The five micrographs are shots have been made. The five micrographs are later examined on a light box and several separated later examined on a light box and several separated points are located on the two films that are closest points are located on the two films that are closest to focus. The smallest distances between two points to focus. The smallest distances between two points are located on both negatives and converted into are located on both negatives and converted into actual nanometer distances based on an accurate actual nanometer distances based on an accurate knowledge of the magnification (Figure 6.55). It is knowledge of the magnification (Figure 6.55). It is important to locate the points on two films, since important to locate the points on two films, since electron noise ("snowy" background) may give an electron noise ("snowy" background) may give an impression of two points that do not physically exist. impression of two points that do not physically exist. A through-focus series may be used with some A through-focus series may be used with some specimens in order to obtain the most accurate specimens in order to obtain the most accurate focus with the highest possible resolution.focus with the highest possible resolution.

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Figure 6.55Resolution standard, evaporated Pt/Ir film showing aresolution of 0.4 nm at points circled. Magnification bar= 50 nm.

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ResolutionResolution The The lattice testlattice test is based on demonstrating a crystallin is based on demonstrating a crystalline lattice of known spacings. Simply stated, if one sees e lattice of known spacings. Simply stated, if one sees the lattice structure in a graphitized carbon particle or the lattice structure in a graphitized carbon particle or a single crystal gold foil (Figure 6.56), then distances oa single crystal gold foil (Figure 6.56), then distances of 0.34 nm and 0.20 nm, respectively, are being resolvef 0.34 nm and 0.20 nm, respectively, are being resolved. There are several problems with taking such figures d. There are several problems with taking such figures as being strictly accurate, since astigmatism and electas being strictly accurate, since astigmatism and electron noise may artificially enhance the resolving capabron noise may artificially enhance the resolving capabilities supposedly being demonstrated. Most microscoilities supposedly being demonstrated. Most microscope manufacturers will provide two figures for resolvinpe manufacturers will provide two figures for resolving power, for example, lattice = 0.14 nm and point-to-pg power, for example, lattice = 0.14 nm and point-to-point = 0.30 nm. oint = 0.30 nm.

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ResolutionResolution The The Fresnel fringe methodFresnel fringe method (Reisner, 1956) (Figures 6.57 and 6.58) (Reisner, 1956) (Figures 6.57 and 6.58) for calibrating resolution is undoubtedly the most convenient mfor calibrating resolution is undoubtedly the most convenient method since the test specimen, a holey film, is readily available to ethod since the test specimen, a holey film, is readily available to all microscopists. After correcting for astigmatism, the fringe is foall microscopists. After correcting for astigmatism, the fringe is focused as accurately as possible and a series of micrographs is takcused as accurately as possible and a series of micrographs is taken by varying the focus slightly between the various exposures. Ten by varying the focus slightly between the various exposures. The films are examined for that negative that shows the finest frinhe films are examined for that negative that shows the finest fringe by being slightly overfocused. The fringe width is then evaluatge by being slightly overfocused. The fringe width is then evaluated by measuring the distance from the center of the overfocus fried by measuring the distance from the center of the overfocus fringe (e.g., the dark line on the negative) to the center of the white nge (e.g., the dark line on the negative) to the center of the white space just inside of the dark line (Figure 6.57B). This distance in nspace just inside of the dark line (Figure 6.57B). This distance in nanometers is the resolving power of the instrument. Of course, oanometers is the resolving power of the instrument. Of course, one must have a good magnification calibration in order to deterne must have a good magnification calibration in order to determine the fringe width accurately. Since this method is based on amine the fringe width accurately. Since this method is based on accurate stigmation and focusing of the microscope, an error of 20ccurate stigmation and focusing of the microscope, an error of 20% is not unusual. However, with experience this simple method % is not unusual. However, with experience this simple method may prove adequate for all but the most precise situations. may prove adequate for all but the most precise situations.

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Figure 6.56Resolution standard, gold foil. The lattice spacingsshow a resolution better than 0.204 nm. Final magnificationof print is 2.4 million times.

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Figure 6.57(A) Convenient resolution standard, Fresnel fringemethod. The fringe width is measured, based on knownmagnification, and the finest fringe width measuredis equal to resolution. (B) Enlargement showing distanceto be measured indicated by arrowheads.

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Figure 6.58Positive print showing edge of holey film with Fresnelfringe (white line, arrow) in overfocused condition.

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Levels of Usage of the Levels of Usage of the Transmission Electron Transmission Electron

MicroscopeMicroscope The preceding chapter was directed toward giThe preceding chapter was directed toward giving the investigator a sound background in thving the investigator a sound background in the fundamentals of the transmission electron me fundamentals of the transmission electron microscope. For serious, committed electron miicroscope. For serious, committed electron microscopists maintaining their own equipment, croscopists maintaining their own equipment, this level of detail—and even much more—is a this level of detail—and even much more—is a necessity. However, there are several other levnecessity. However, there are several other levels of experience that might be appropriate for els of experience that might be appropriate for individuals using an electron microscope. individuals using an electron microscope.

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MicroscopeMicroscope The experienced user and maintainer.The experienced user and maintainer. This is an This is an

individual who has responsibility for the operation individual who has responsibility for the operation and maintenance of the microscope. Considerable and maintenance of the microscope. Considerable experience in the theoretical basis of microscopy, experience in the theoretical basis of microscopy, electronics, and a mechanical inclination are electronics, and a mechanical inclination are necessary. If the microscope fails, and even new necessary. If the microscope fails, and even new ones do fail, this individual must have the ones do fail, this individual must have the capability to repair it. Rarely do electron capability to repair it. Rarely do electron microscope users have full maintenance microscope users have full maintenance capability. If the microscope breaks down or if capability. If the microscope breaks down or if specialized replacement parts are needed, then specialized replacement parts are needed, then service representatives may be called upon. service representatives may be called upon.

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MicroscopeMicroscopeThe experienced user with a maintenancThe experienced user with a maintenance contract.e contract. Such researchers are involved Such researchers are involved in the routine operation of the microscopin the routine operation of the microscope and the recording of images. The level e and the recording of images. The level of familiarity with the instrument may inof familiarity with the instrument may include routine alignment and perhaps rouclude routine alignment and perhaps routine servicing (cleaning of apertures and tine servicing (cleaning of apertures and specimen anticontaminators, replacemespecimen anticontaminators, replacement of burnt-out filaments, calibration of nt of burnt-out filaments, calibration of magnification and resolving power, etc.). magnification and resolving power, etc.).

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MicroscopeMicroscope Major repairs and routine cleaning of sensitive microsMajor repairs and routine cleaning of sensitive microscope parts (polepieces, specimen stages, column linercope parts (polepieces, specimen stages, column liners, etc.) may be left to service engineers as part of an as, etc.) may be left to service engineers as part of an annual service contract. Such yearly contracts may varnnual service contract. Such yearly contracts may vary from 5 to 10% of the original cost of the instrument. Iy from 5 to 10% of the original cost of the instrument. In situations where productivity cannot be interrupted n situations where productivity cannot be interrupted by a researcher becoming involved in microscope maiby a researcher becoming involved in microscope maintenance, such contracts are probably quite cost effecntenance, such contracts are probably quite cost effective. In addition, the contracts may include the replactive. In addition, the contracts may include the replacement of extremely expensive parts (high voltage tankement of extremely expensive parts (high voltage tanks, vacuum system components, electronic circuit boars, vacuum system components, electronic circuit boards, stages and specimen holders, etc). ds, stages and specimen holders, etc).

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MicroscopeMicroscope The assisted viewer.The assisted viewer. Individuals who have only a temp Individuals who have only a temporary need for electron microscopy may rely on more orary need for electron microscopy may rely on more experienced personnel to assist them in specimen preexperienced personnel to assist them in specimen preparation and the viewing of specimens in the electron paration and the viewing of specimens in the electron microscope. Often, researchers in need of electron mimicroscope. Often, researchers in need of electron microscopy will contact a microscopist at their own or pcroscopy will contact a microscopist at their own or perhaps at nearby institutions to arrange for the needeerhaps at nearby institutions to arrange for the needed services. In this case, the researcher may simply vied services. In this case, the researcher may simply view the specimens while a trained individual oversees tw the specimens while a trained individual oversees the operation of the microscope. With time, and a contihe operation of the microscope. With time, and a continued need for electron microscopy, the researcher manued need for electron microscopy, the researcher may opt to become more personally involved in the opery opt to become more personally involved in the operational procedures. However, a more efficient use of tational procedures. However, a more efficient use of the researcher's time may still involve assistance by an he researcher's time may still involve assistance by an experienced microscopist.experienced microscopist.

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MicroscopeMicroscope A user contracting for service.A user contracting for service. Occasionally a researcher Occasionally a researcher

may need electron microscopy as a small part of a study, may need electron microscopy as a small part of a study, perhaps to confirm some data obtained by other perhaps to confirm some data obtained by other techniques. The researcher may have little experience in techniques. The researcher may have little experience in image interpretation and no experience or interest in image interpretation and no experience or interest in learning even the basics of microscope operation. Such learning even the basics of microscope operation. Such individuals usually contact individuals usually contact consultantsconsultants who are experienced who are experienced in the area of research in which the investigator is involved in the area of research in which the investigator is involved and who have access to an electron microscope. The and who have access to an electron microscope. The researcher may send specimens to the consultant, who researcher may send specimens to the consultant, who then studies the specimens in the electron microscope, then studies the specimens in the electron microscope, records images, and provides the researcher with records images, and provides the researcher with photographic prints and perhaps even an analysis of the photographic prints and perhaps even an analysis of the micrographs. Such consultants generally command micrographs. Such consultants generally command premium fees since they are experienced in both electron premium fees since they are experienced in both electron microscopy as well as the interpretation of the images. microscopy as well as the interpretation of the images.

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Shared FacilitiesShared Facilities Due to the high cost of acquiring and maintaining an electron miDue to the high cost of acquiring and maintaining an electron microscope and associated support equipment, shared facilities arcroscope and associated support equipment, shared facilities are becoming more common than individual researchers having sue becoming more common than individual researchers having such units. A shared or centralized facility may involve only one elech units. A shared or centralized facility may involve only one electron microscope serving several researchers with similar researctron microscope serving several researchers with similar research interests or it may be quite extensive with many electron micrch interests or it may be quite extensive with many electron microscopes equipped for various specialized needs. Users of such faoscopes equipped for various specialized needs. Users of such facilities may be operating at different levels of experience, so that cilities may be operating at different levels of experience, so that trained microscopists and technologists may be involved in variotrained microscopists and technologists may be involved in various service and training activities. Central facilities are usually finaus service and training activities. Central facilities are usually financially supported to various extents by a central administration ancially supported to various extents by a central administration and/or by a system of external grants and contracts. Often there is nd/or by a system of external grants and contracts. Often there is an established fee structure for the various levels of usage of the an established fee structure for the various levels of usage of the facility. facility.

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Microscopy 2009 Wk14 TEMd