Fluorescence and electron microscopy

Microscopy

T.Y.B.Sc. (Biotechnology)Paper IIIUnit III

created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES

College of Arts, Science and Commerce, Mumbai, INDIA

created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

Fluorescence Microscopy


Why do we need fluorescence microscopy?

Gabi Barmettler DiOC6(3) Fluorescence Staining and Phase Contrast Imaging of MDCK Cells


• Better resolution• Better identification of specific intracellular

components• Better understanding of molecular

interactions

Why do we need fluorescence microscopy?


Principle• The fluorescence microscope depends on two

intrinsic properties of the substance to be observed– FLUORESCENCE– PHOSPHORESCENCE


FLUORESCENCE• Fluorescence is the emission of light by a

substance that has absorbed light or other electromagnetic radiation.

• Emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation.– STOKE’S SHIFT


FLUORESCENCE- STOKE’S SHIFT

• Stoke’s shift is the difference (in wavelength or frequency units) between positions of the band maxima of the absorption and emission spectra


Why does Stoke’s shift occur?• Energy levels

o Ground state (no light absorbed)o Excited state (light energy

absorbed)

• Each energy level is divided intoo Vibrational Energy levelo Rotational Energy Levelo Heat Energy level

• Non-radiative loss of energy• Remaining energy lost as

fluorescent light as electron comes down to ground state.

Jablownski Diagram


PHOSPHORESCENCE• Phosphorescence is a specific

type of photoluminescence in which a phosphorescent material does not immediately re-emit the radiation it absorbs. The slower time scales of the re-emission are associated with "forbidden" energy state transitions in quantum mechanics. As these transitions occur very slowly in certain materials, absorbed radiation may be re-emitted at a lower intensity for up to several hours after the original excitation.


The Technique• Fluorophores: Molecules that have a

conformation that allows fluorescent emission.• Intrinsic Fluorophores: Fluorescent molecules

inherent to the sample.E.g. DNA, Protein (Trp)

• External fluorophore: Added to sample to label certain specific component of the sample.E.g. Green Fluorescent protein, Fluorescein


Image of artery walls using intrinsic fluorophore - elastin

Neuron stained with GFP

HeLA cells showing Anaphase. DNA stained with DAPI. Microtubules stained with Fluorescein Red


Instrumentation


Instrumentation1. Light Source: Xenon Lamp or Mercury Arc Lamp.

Should provide UV and visible light.2. Excitation Filter: Selects the wavelength of light

absorbed by fluorophore.3. Dichroic Mirror: Reflects the light coming from

light source and transmits the light coming from specimen.

4. Lens System: objective and ocular lens.5. Emission filter: Allows only the emitted light to

pass through.• At one time emission filter allows only a single

wavelength of light to pass through, so only a single colour image is obtained at a time.


Applications1. Non-specific dye binding2. Immunofluorescence3. GFP-tagging


Non-specific Dye Binding• Fluorescent dyes

bind to specific kind of molecules– DNA- Ethidium

Bromide (not used in microscopy), Hoechst Stain (absorbs UV light and fluoresces blue). Fibroblast cell line stained with Hoechst

33342 nucleic acid stain.


Immunofluorescence

DNA-HoechstMitochondria- Mitotracker RedJunction proteins- fluorescent antibodies.


GFP- tagging• Green Fluorescent Protein from

Aequorea victoria.• Can be fused to any gene

(recombinant DNA technology), thereby generating a recombinant protein that fluoresces green.

• Advantage- Recombinant protein in cell will fluoresce without any staining, thus live cells can be image.


Modified GFP• By changing amino acid sequence of

GFP new proteins made-– RFP = Red– YFP = Yellow– CFP = Cyan


Limitations of Fluorescence Microscopy

1. Fluorophore used might interfere with metabolic pathway studied. E.g. GFP is a large protein and might affect movement of tagged protein.

2. Excitation light might damage live tissue3. Excited fluorophore might react with oxygen and

generate free radicals toxic to cell.4. Photobleaching – While in excited state fluorophore

might undergo covalent modification that destroys their ability to fluoresce.


CONFOCAL MICROSCOPY


Principle• Pin-point illumination of the specific portion

of the specimen to be observed.• Pin-hole in front of detector blocks out-of-

focus light.• Images have a higher resolution.

Image of mouse intestinal wall

Wide-field Confocal


Instrumentation1. Light Source – Zirconium Arc

Lamp or Laser Light Source2. Scanning Motors- Vertically and

horizontally scanning mirrors allow changing the focal point laterally and horizontally, thus collecting image from the entire specimen, one point at a time.

3. Objective Lens- focuses the fluorescent light from each point on specimen to detector pin-hole

4. Dichroic Mirror5. Pin-hole – Eliminates out-of-

focus light. Two types – Light source pin-hole and Detector Pin-hole

6. Detector – Photomultiplier tube


Optical sectioning• Division of 3D specimen

into several 2D focal planes

• Image created by a confocal microscope is a thin planar region of a 3D specimen.

• The 2D image is generated because of the focal plane created.

Sections of a pollen grain


Optical sectioning allows clarity and better visualization


Z-stacking• Data gathered from a series

of optical sections imaged at short and regular intervals along the z axis are used to create a 3D reconstruction.

• This compilation of a linear array of 2D sections to obtain a 3D model of the specimen is called Z-stacking


3D image made by Z-stacking


Applications1. Imaging highly expressed molecules.2. Protein interactions within the cell3. To study 3D architecture of cells4. Enables visualization of specific sections of

cell.


Electron MicroscopyTransmission Electron Microscopy (TEM)

Scanning Electron Microscopy (SEM)

Breast Cancer Cell on SEM

TEM image of rat liver nucleus


Difference from Light MicroscopeLight Microscope Electron Microscope

Illumination Visible Light ElectronsIllumination Point

Bottom of microscope Top of microscope

Illumination Source

Lamp/ Natural Light Tungsten filamnet

Lens System Glass Lenses Electrical CoilsLenses (i) Condensor (i) Condensor

(ii) Objective (ii) Objective(iii) Eye piece (iii) Projector

Visualization Eye Fluorescent Screen or photographic film


Difference from Light Microscope


Properties of Electrons

• Electron are negatively charged sub-atomic particles

• Electron given enough energy leave the atom and fly off in a stream.

• Tungsten is used as a source of electrons


Interaction of Electrons with Matter

• EM maintained in vacuum because air can absorb electrons.

• Interaction with specimen leads to generation of many different types of rays:1. Transmitted Electrons2. Elastically scattered electrons3. Inelastically scattered electrons4. Back-scattered electron5. Secondary electrons 6. Visible light and X-rays


Instrumentation1. Electron Gun: Located on the top of the

microscope. Its is a tungsten filament in a negatively biased shield with an aperture.

2. Microscope Column: Evacuated metal tube. All components aligned one on top of the other. Provides shielding from X-rays.

3. Electromagnetic lens or coils: Condenser, objective and projector coils. Each coil is in a hollow metal cylinder. Generates a magnetic field aligned with the electron beam.

4. Transformers: provide high voltage current to the electron gun.

5. Vacuum Pump: Maintain vacuum within the microscope column.

6. Fluorescent Screen: for image capture7. Water Cooling system: Prevents over-

heating.


Working

1. Image Formation2. Magnification3. Resolving Power


Image Formation• Occurs by electron scattering• Dispersed electrons from specimen

converted to visible form on fluorescent screen

• Energy of electrons converted into visible light

• Electrons reaching the screen form bright spots, areas where electrons don’t reach form dark spots

• Electron dense: Areas which scatter electrons

• Electron dispersion is directly proportional to atomic number of atom dispersing.

• Higher atomic number better dispersion.• Biological samples have low atomic

numbers. Thus stained with salts of high atomic number elements.


Magnification• Objective and Projector coils

responsible for magnification.• Intermediate coils can be fitted

to increase magnification.• Total magnification = product of

magnification by individual coils.• Eg. If projector = 200X and

objective = 100 X • Total magnification = 20000X• Highest magnification achieved –

1,000,000X

E. coli at 1000X

E. coli at 1000000X


Resolving Power

• Limited by wavelength of illuminating electron bean.

• Limit of resolution = half the wavelength• If λ= 0.037 Ao then D (limit of resolution) = 0.018 Ao

• Practically, best resolution achieved is 4 – 10 Ao


TEM

• A beam of electrons is transmitted through an ultra thin specimen

• Sample preparation is different, sample should allow electrons to pass through

TEM image of eukaryotic cell


SEM

• Image formation is because of secondary and back-scattered electrons

• Gives 3-D architecture of specimen

SEM of eukaryotic cell


Sample Preparation

• For SEM – Staining– Sectioning by microtome

• For TEM- Freeze fracture– Staining


Staining

• Two standard methods• Used for both TEM and SEM– Shadow Casting– Negative Staining


Shadow Casting• A technique used to improve

contrast.1. Sample on copper grid is placed

in evacuated chamber2. Heavy metal like chromium,

palladium, platinum or uranium is evaporated at an angle from a filament

3. As the metal gets deposited at an angle it piles up on the side from which it is deposited while the other side remains clear.

4. In the EM, the areas with stain show dark while areas with no stain appear bright.

Shadow casting heightens the profile and adds depth to the image.


Negative Staining

1. Involves treatment of material with phosphotungstic acid

2. The stain penetrates the empty spaces of the cell

3. When material is washed and studied under the microscope, it shows light areas of the material while interstices filled with stain appear dark.


Sectioning sample for SEM• Sections are cut by glass or

diamond knives• Section have to be thin enough to

allow electrons to pass• Glass knives sharp but fragile so

diamond knives used.• Instrument – Microtome• Regular microtome – 500 nm thick

section• Ultra microtome – 10-50 nm thick

sections• Sections once cut are floated on

acetone water and picked up on perforated copper grid.


Freeze Fracture - TEMDone to impart a 3D texture and better

resolution to image.1. Specimen tissue frozen at -130

degrees in liquid freon2. Specimen is transferred to an

evacuated chamber at -100 degrees3. Microtome used for cracking or

fracturing tissue.4. Fractured sample is left in vacuum

or removed in water5. Specimen is then subjected to

shadow casting

Devices & Hardware

Fluorescence and electron microscopy