Course: PG Pathshala-Biophysics

Paper 10 : Techniques Used In Molecular Biophysics II (Based on Spectroscopy)

Module: M3. Transmission Electron Microscope

Content Writer : Dr. M. R. Rajeswari, AIIMS, New Delhi

Learning Objectives:

The present module is designed to provide the biophysical principle, instrumentation and

applications of ‘transmission electron microscope (TEM)’. There are following learning

objectives for elucidation of in-depth knowledge of TEM.

1. Principle of Transmission electron microscope.

2. Function & Limitation of TEM

3. Biophysical principle of TEM

4. Biological sample preparation for TEM

Introduction of Transmission electron microscope (TEM):

The transmission electron microscope is a type of electron microscope in which a high energy

beam of electrons is irradiated on a thin specimen, and the electrons passed through the

specimen which results in the formation of an image. In general, the TEM has the same basic

principle as of the light microscope. However, instead of light rays the electrons beam is

utilized to produce image of the object and magnification is achieved by electromagnetic fields.

TEM has been utilized as an analytical/visualizing method especially in the physical/ materials

sciences, and biological/medical sciences with the specific applications in cancer biology,

bacteriology, virology, nanotechnology etc.

Ernst Ruska and Max Knoll in 1931 constructed the first transmission electron microscope for

which, Ruska got the Nobel Prize (in physics) in 1986 for the development of transmission

electron microscopy. However, the first electron micrograph of eukaryotic cells was taken by

Keith Porter in 1940s and after that due to TEM it is made possible to study nanoscale

structures like cells and tissues.

1. Transmission electron microscope

2. To know the working principle of transmission electron microscope

3. To elucidate the instrumentation and specimen preparation for transmission electron

microscopy.

4. To explore the application of TEM in infectious biology, cancer research, material

science and nanotechnology.

Introduction:

You have learnt about the light microscopy in the previous module 2. Resolution of a

microscope is primarily based on the wavelength of illumination source which sets a limit on

how small an object be seen. Electrons have 100,000 times shorter wavelength than visible

light (which is being utilized in conventional light microscope). This property of shorter

wavelength has been utilized in the field of microscopy to improve the limit of resolution/

resolving power and that laid the foundation of Electron microscopy. In general, a beam of

electrons is utilized to illuminate the specimen in an electron microscope which ultimately

creates an image. The improvement in terms of resolution may be seen by the fact that an

electron microscope can show a much higher resolution of 0.2nm and magnifications upto

2,000,000 x. as compared to a resolution of 200nm and magnification upto 2000x by a light

microscope.

The electron microscopes are classified into two following types according to the mode of

their function.

Max Knoll and Ernst Ruska in 1931 constructed the first transmission electron microscope. However, the biological samples were analysed in 1940s.

Max Knoll Ernst Ruska

Ernst Ruska got the Nobel Prize (in physics) in 1986 for the development of transmission electron microscopy.

INTRODUCTION

1. Transmission electron microscope (TEM)

2. scanning electron microscope (SEM)

Principle and function:

Transmission electron microscope is similar to optical microscopy, except that the photons are

replaced by electrons. By propelling electrons at a thin sample, and detecting those transmitted

through it, a map of local densities of the sample is obtained. If there are ordered structures

such as crystals are present in the sample then diffraction information can also revealed.

When high voltage is applied, the electron gun gets heated up and emits electrons. The emitted

electrons form a cloud around the tip of filament, and are propelled down the column due to high

potential (40-120kv) generated between the filament and the anode. The electron beam is focused on

to the specimen by means of condenser lens.

The objective lens forms the initial enlarged image of the illuminated portion of the specimen in a

plane that is suitable for further enlargement by the intermediate lens and projector lens. By varying

the current in the objective lens, the image of the specimen is focused. The intermediate and

projector lens project the final magnified image on the screen or photographic film or CCD camera.

The electron microscope provides information in the image in the form of variations of electron

intensity over its area. Transmitted electrons are converted into visible light by a fluorescent screen.

The image seen can be recorded permanently on silver halide coated photographic film. The electron

beam liberated free silver halide, which after chemical treatment (development) produces film

negatives. These negatives can be further processed to get positives on the photographic paper. This

image recorded on the photographic paper is known as an electron micrograph. The image

information can also be recorded in digital format by a CCD camera. Figure 7.28b shows the electron

micrographs of transmission electron microscope in which adverse effects of meropenem

(Carbapenem antibiotic) are seen on a drug susceptible strain of Acinetobacter baumannii.

Limitations of TEM

There are following drawbacks of TEM technique.

1.) Many materials require extensive sample preparation to produce a sample thin enough

to be electron transparent, which makes TEM analysis a relatively time consuming

process with a low throughput of samples.

2.) Graphene, a carbon nanomaterial, relatively transparent, very hard and just one atom

thick, is currently being used as a platform on which the materials to be examined are

placed. Being almost transparent to electrons, a graphene substrate has been able to

show single hydrogen atom and hydrocarbons.

3.) The structure of the sample may also be changed during the preparation process. Also

the field of view is relatively small, raising the possibility that the region analyzed may

not be characteristic of the whole sample.

4.) There is potential that the sample may be damaged by the electron beam, particularly in

the case of biological materials.

Biophysical Principle of TEM:

The basic principle of TEM is similar to the light microscopy (optical microscope).

Theoretically, as seen in light microscopy, the limit of resolution (d) of a microscope is the

smallest distance between 2 points that can be seen using a microscope. This is a measure of

the clarity of the image.

The limit of resolution can be limited by the wavelength (λ) of the photons that are being used

to probe the sample, and the numerical aperture (NA).

d = 0.61λ/nsinα= λ/2NA (1)

n sinα = numerical aperture (NA) (2)

where, n is the refractive index of the medium in which the lens is working and α is the

maximum half-angle of the cone of light that can enter the lens (measure of light-gathering

ability).

As we know that wavelength of visible light in between 400–700 nm. Refractive index of air=

1; and maximum sin α = 90 i.e. 1, therefore, no lens working in air can have a numerical

aperture value greater than 1.

NA = 0.95 (max. with air) and maximum (~1.5) when oil-immersion objective lens are used.

By all these above values, it has been estimated that maximum resolution of an optical

Microscope is 250 nm. To increase the resolution, we have to use less value of wavelength

(λ) which can be done with the help of electrons.

Electrons are negatively charged; very small particles and they behave like wave as well as

particle (de Broglie phenomenon). In TEM an electron's velocity approaches the speed of light,

c. Due to their wave-like properties, electrons behave like a beam of electromagnetic radiations

and the wavelength of electrons is related to their kinetic energy (de Broglie equation).

where, h is Planck's constant, m0 is the rest mass of an electron and E is the energy of the

accelerated electron.

Electrons beams are emitted by heating of a tungsten filament. The acceleration of electrons is

done by an electric potential (voltage, measured in volts) in the anode. A higher anode voltage

gives a higher speed to the electrons and also the electrons have a smaller de Broglie

wavelength (λ = h/mv). Therefore, faster the electrons travel, the shorter their wavelength. As

the wavelength is reduced, the resolution is increased. Therefore, the resolution of the

microscope is increased if the accelerating voltage of the electron beam is increased.

As the wavelength of electrons is much shorter than photons of light, the resolution attainable

for TEM images is much higher than that from a light microscope. Therefore, from TEM image

we can reveal the finest details of internal structures of cells.

Working principle of TEM: In TEM, a beam of electrons of very short wavelength is emitted

from a tungsten filament at the top of a cylindrical column. The TEM system has vacuum

because if air present in the column, it creates the collision with electrons and hence the

scattering of electrons are avoided. Along the column, at specific intervals magnetic coils are

placed which acts electromagnetic condenser lens and are required to focus the electron beam.

The thin specimen stained with an electron dense material is placed at the stage in the vacuum.

The focused beam of electron then transmitted through this thin specimen and gets scattered by

the internal structures. The transmitted electrons carries information about the structure of the

specimen through spatial variation and this information in the form of an "image" is then

magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent

screen/photographic plate, or light sensitive charge-coupled device(CCD) camera. The image

detected by the CCD may be displayed in real time on a monitor or computer.

Instrumentation in TEM: Following are the main components which are required for the

overall functioning of TEM (Figure 1).

1. Emission source/ Electron gun: The initial/first component of a TEM is an electron gun

which is mounted at top of the microscope (Figure 1). An electron gun emits the intense beam

of electrons into the cylindrical vacuum chamber which accelerates the between the cathode

and the anode. Tungsten filament or lanthanum hexaboride are generally used materials which

are utilized as electron gun source. In general, thermionic electron gun and field emission gun

are main types of electron gun. In overall mechanism of electron emission, loosely bound

valence electrons are utilized. However, these electrons cannot escape from the metal surface

as positively charged nucleus always tries to pull back the free electrons. Electrons have to rise

above the potential barrier for escaping from the surface of the metals and the energy required

to overcome this potential barrier is known as work function (φ).

2. Optics: the optical system in TEM has a function to converge the electron beam. The

magnification also can be modulated by simply altering the current flowing through

electromagnetic coils. Lens system of TEM can be divided into three levels condenser,

objective and projector lenses. Condenser lens system works to form primary beam and

objective lenses focus the beam that comes through the sample. Finally the projector lenses

expand beam onto phosphorus screen. When a beam of light (or electrons) encounters a

specimen, the specimen alters the physical characteristics of illuminating beam.

Figure1. Organization of Transmission electron microscope. (Source: By Gringer (talk) -

Commons: Scheme TEM en.png, CC BY-SA 3.0,

https://commons.wikimedia.org/w/index.php?curid=5624170

3. Imaging systems: The imaging system also contains the electromagnetic lens system and a

screen with phosphorescent plate. The sample holder (mechanical arm) holds the specimen and

the beam of electrons are focused on the specimen by the condenser which consists of

electromagnets called magnetic lenses. The plate glows when hit by the electrons after passing

through the specimen. A phosphor screen is made up of 10-100 μm fine zinc sulphide alone or

coupled with CCDs is used to visualize this final image.

Biological sample preparation for TEM:

Sample preparation is most crucial aspect for TEM. There are following various steps in

sample preparation.

1) Primary Fixation: It is a process to preserve the sample and prevention from degradation.

Glutaraldehyde is a general fixative in TEM by which the protein molecules of biological

sample gets covalently cross linked to their neighbours.

2) Osmium tetroxide (OsO4) fixation: Osmium tetroxide (OsO4) fixation acts as a secondary

fixation. The OsO4 fixative (binds with the phospholipid head regions of membrane and creates

a contrast with the neighbouring cytoplasm. It also helps in the stabilization of many proteins

by transforming them into gels without destroying the structural features. Tissue proteins,

which are stabilized by OsO4 and does not coagulate by alcohols during dehydration. Heavy

metals like uranium and lead are also used to increase electron density to the internal

structures.

3) Dehydration: This step is done to remove water content form the tissue sample and replaced

with an organic solvent.

4) Infiltration, Embedding and Polymerization: Epoxy resins are utilized to penetrate the cells

which fill the space and give hard structure to tolerate the pressure of cutting. After processing

the embedding done using flat molds. Next is polymerization step in which the resin is allowed

to set overnight at a temperature of 60 degree in an oven.

5) Sectioning: The polymerized specimen cut into very thin slices (30-60 nm sections) using an

ultramicrotome. These sections are then collected onto a copper grid and viewed under the

microscope.

APPLICATIONS OF TEM

Transmission electron microscopy finds a lot of applications in the area of infectious biology

e.g. mechanism of pathogenesis of bacteria, viruses and fungi, diagnosis and therapeutics of

infections, cancer biology, Nano-biotechnology etc.

In infectious biology: Prokaryotes are the smallest unicellular organism which can’t be

visualized through naked eye. Light microscopes can decipher the morphology and certain

organelle of prokaryotic cells. In beginning, researchers have defined prokaryotic cell via using

light microscope of 1.4 numerical apertures and 0.4 µm resolution. However improved

structure with high resolution is possible only through electron microscope. The following

figures 2 and Figure 3 show the electron micrograph of variola orthopox virus inside tissue and

Methicillin resistant Staphylococcus aureus ingestion by neutrophil, respectively, which can

never be seen by optical microscope. Therefore, it leads to the identification of causative

organism and its precise location of the infection which is crucial in diagnostics and

therapeutics in Infectious biology.

Figure2: Smallpox Tissue section containing variola orthopox viruses TEM PHIL 2291 lores.

(source:https://commons.wikimedia.org/wiki/File:Smallpox_Tissue_section_containing_variol

a_orthopox_viruses_TEM_PHIL_2291_lores.JPG)

Figure3. Methicillin resistant Staphylococcus aureus ingestion by neutrophil.

Source:https://commons.wikimedia.org/wiki/File:MRSA,_Ingestion_by_Neutrophil.jpg

TEM In cancer biology: Cancer diagnostic approaches widely use TEM to understand poorly

differentiated carcinomas, hematopoietic and lymphoreticular malignancies by identification of

cellular morphology prior to the beginning of therapeutics. For example in bone marrow biopsy

samples suspected of myelodysplastic syndrome, pathologic changes can be easily discerned

with the help of microscopy (Figure 4).

Figure4. Macrophages killing cancer cells.

(source:https://commons.wikimedia.org/wiki/File:Macs_killing_cancer_cell.jpg)

Application of TEM in Nano-biotechnology:

The TEM has the ability to determine the positions of atoms within materials which has made

an indispensable tool for nano-technologies research. Transmission electron microscopy is

generally utilized in the field of nanotechnology for exploration of structural information at the

atomic level. The focused beam of electrons travels through the specimen which is in general a

nano-particle (e.g. gold nano particle; GNP). Depending on the density of the nano-material

present, electrons are transmitted through the sample, however some of the electrons get

scattered and disappear from the beam. At the bottom of the microscope the

transmitted/unscattered electrons hit a fluorescent screen, which gives rise to a "shadow image"

of the specimen with its different parts displayed in varied darkness according to their density.

Figure 5 shows the application of TEM microscopy to see the morphology and size

determination of lab-synthesized gold nano-particle. Electron micrograph confirms the

morphology as (anisotropic) and average size of GNP was found 40nm.

Fig.5: Tem Micrograph of Gold nano-particles at different resolution a) 100 nm resolution and

b) at 50nm resolution.TEM micrograph confirms the morphology and size of GNP. Average

size of GNP was found 40nm with irregular shape (Courtesy: Image given by Dr. Abhishek

Chaudhary; nano-biotechnology research work).

Summary:

Advantage of TEM over conventional microscopy is its capability of producing images with

higher resolution.

Efficiency of TEM can be accessed at atomic levels and higher resolution power has enabled

modulation in electronic and chemical structure, crystal orientation and temporal imaging.

TEM works on the same basic principles as the light microscope but uses electrons instead of

light (lanthanum hexaboride (LaB6) single crystal, tungsten filament, etc., are used as electron

source). Much lower wavelength of electrons makes it possible to get a resolution thousand

times better than with a light microscope.

The beam of electron is being modulated by magnetic field to generate magnetic lenses with

varying power. Additionally, electrostatic field can also cause deflection of electron at various

angles.

As the beam of electron passes through the sample which is approximately 100nm thick, image

thus formed is sent to an imaging device (fluorescent/ photographic film or charge-coupled

device) for magnification.

In the life sciences, the specimen preparation is a crucial step and it also limits the resolution in

the electron microscope.

Transmission electron microscopy has provided wide applications in the area of infectious

biology which include the mechanism of pathogenesis of bacteria, viruses and fungi, diagnosis

and therapeutics of infections.

Cancer diagnostic approaches widely use TEM to understand poorly differentiated carcinomas,

hematopoietic and lymphoreticular malignancies by identification of cellular morphology prior

to the beginning of therapeutics

The TEM has the ability to determine the positions of atoms within materials which has

made an indispensable tool for nano-technologies research.