Fluorescence and electron microscopy
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Transcript of 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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Why do we need fluorescence microscopy?
Gabi Barmettler DiOC6(3) Fluorescence Staining and Phase Contrast Imaging of MDCK Cells
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
• Better resolution• Better identification of specific intracellular
components• Better understanding of molecular
interactions
Why do we need fluorescence microscopy?
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Principle• The fluorescence microscope depends on two
intrinsic properties of the substance to be observed– FLUORESCENCE– PHOSPHORESCENCE
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Instrumentation
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Applications1. Non-specific dye binding2. Immunofluorescence3. GFP-tagging
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Immunofluorescence
DNA-HoechstMitochondria- Mitotracker RedJunction proteins- fluorescent antibodies.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Modified GFP• By changing amino acid sequence of
GFP new proteins made-– RFP = Red– YFP = Yellow– CFP = Cyan
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
CONFOCAL MICROSCOPY
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Optical sectioning allows clarity and better visualization
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
3D image made by Z-stacking
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Applications1. Imaging highly expressed molecules.2. Protein interactions within the cell3. To study 3D architecture of cells4. Enables visualization of specific sections of
cell.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Electron MicroscopyTransmission Electron Microscopy (TEM)
Scanning Electron Microscopy (SEM)
Breast Cancer Cell on SEM
TEM image of rat liver nucleus
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Difference from Light Microscope
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Working
1. Image Formation2. Magnification3. Resolving Power
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
SEM
• Image formation is because of secondary and back-scattered electrons
• Gives 3-D architecture of specimen
SEM of eukaryotic cell
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Sample Preparation
• For SEM – Staining– Sectioning by microtome
• For TEM- Freeze fracture– Staining
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
Staining
• Two standard methods• Used for both TEM and SEM– Shadow Casting– Negative Staining
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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.
created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA
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