Fluorescence and electron microscopy
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Principle and basic instrumentation of advanced microscopes like fluorescence, confocal microscopes and Electron microscopes
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Fluorescence and electron microscopy
- 1. Microscopy T.Y.B.Sc. (Biotechnology) Paper III Unit III created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 2. Fluorescence Microscopy created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 3. 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,
- 4. • 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,
- 5. 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,
- 6. 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,
- 7. 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,
- 8. 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 into o Vibrational Energy level o Rotational Energy Level o Heat Energy level • Non-radiative loss of energy • Remaining energy lost as fluorescent light as electron comes down to ground state. Jablownski Diagramcreated by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 9. 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,
- 10. 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,
- 11. 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,
- 12. Instrumentation created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 13. Instrumentation 1. 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,
- 14. Applications 1. Non-specific dye binding 2. Immunofluorescence 3. GFP-tagging created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 15. 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,
- 16. Immunofluorescence DNA-Hoechst Mitochondria- Mitotracker Red Junction proteins- fluorescent antibodies.created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 17. 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,
- 18. 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,
- 19. 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 tissue 3. 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,
- 20. CONFOCAL MICROSCOPY created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 21. 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,
- 22. Instrumentation 1. Light Source – Zirconium Arc Lamp or Laser Light Source 2. 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 Mirror 5. 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,
- 23. 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,
- 24. Optical sectioning allows clarity and better visualization created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 25. 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,
- 26. 3D image made by Z-stacking created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 27. Applications 1. Imaging highly expressed molecules. 2. Protein interactions within the cell 3. To study 3D architecture of cells 4. Enables visualization of specific sections of cell. created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 28. Electron Microscopy Transmission 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,
- 29. Difference from Light Microscope Light Microscope Electron Microscope Illumination Visible Light Electrons Illumination Point Bottom of microscope Top of microscope Illumination Source Lamp/ Natural Light Tungsten filamnet Lens System Glass Lenses Electrical Coils Lenses (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,
- 30. Difference from Light Microscope created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 31. 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,
- 32. 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 Electrons 2. Elastically scattered electrons 3. Inelastically scattered electrons 4. Back-scattered electron 5. Secondary electrons 6. Visible light and X-rays created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 33. Instrumentation 1. 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 capture 7. Water Cooling system: Prevents over- heating. created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 34. Working 1. Image Formation 2. Magnification 3. Resolving Power created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 35. 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,
- 36. 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 1000000Xcreated by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
- 37. 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,
- 38. 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,
- 39. 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,
- 40. 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,
- 41. 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,
- 42. Shadow Casting • A technique used to improve contrast. 1. Sample on copper grid is placed in evacuated chamber 2. 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,
- 43. 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,
- 44. 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,
- 45. Freeze Fracture - TEM Done to impart a 3D texture and better resolution to image. 1. Specimen tissue frozen at -130 degrees in liquid freon 2. Specimen is transferred to an evacuated chamber at -100 degrees 3. Microtome used for cracking or fracturing tissue. 4. Fractured sample is left in vacuum or removed in water 5. Specimen is then subjected to shadow casting created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce,
Editor's Notes
- Molecules have various states called energy levels. The electron in a non-fluorescent or undisturbed molecule are said to be in the ground state. When these electrons absorb energy of a photon ( Light Absorption) they jump to a higher energy level called excited state. Each energy level has various electronic, vibrational and rotational energy levels. The excited electron loses some of its energy as: Vibrational energy, Rotational Energy and Heat Energy. All these clubbed together are Non-radiative transfer of energy.
- In case of phosphorescence, the excited electron undergoes an unusual intersystem crossing into another energy state termed as the triplet state. As a result the energy gets trapped in the triplet state. As the movement of electron from singlet excited state to triplet state is thermodynamically unfavoured, the process is slow and considerable energy is lost. Phosphorescent emission thus takes longer than fluorescence and has less energy or longer wavelength.
- Different fluorophores absorb different wavelength of light and have different emission wavelength. Therefore they confer different colours to the tagged samples. Thus by tagging different parts of the cell with different fluorophores, a multicoloured image of the cell can be observed.
- To obtain multicolured images, different fluorophores are excited one at a time and separately imaged. The images are them overlaid to get the final image.
- Used to study the spatial distribution of a specific protein in the specimen. This specificity is achieved by using an antibody against the protein of interest. Then an anti-antibody to the primary antibody against the protein of interest is added. This anti-antibody is tagged with a fluorophore. Thus the fluorophore gets localized to where the specific protein of interest is present. Disadvantage- for allowing the fluorescently labelled antibody to enter, the cell needs to be fixed and permeabilized. This implies, live imaging is not possible.
- Made by Marvin Minsky to overcome the limitations of fluorescence microscope In a conventional wide-filed microscope the entire specimen is flooded with excitation light. Results in fluorescence of the entire specimen, reducing resolution and localized observation. In confocal microscopy, light produced by fluorescence very close to focal plane can be detected. However, the light from part of sample not being visualized is removed so signal intensity is much lower than fluorescence microscope.
- The confocal microscope incorporates the ideas of point-by-point illumination of spceimen and rejection of out-of-focus light. Light source pin-hole allows pin-point focussing of excitation light onto the sample. Detector pin-hole allows only light from focal point to pass through the detector. Thus increasing contrast. The detector is a photomultiplier tube that amplifies the fluorescent signal coming from specimen. In a confocal microscope there is never a complete image of the specimen because at any instant only one point is observed. Thus, for visualization the detector is attached to a computer which builds an image. One pixel at a time.
- Images generated by confocal have much higher contrast and clarity as compared to fluorescence microscopy. Thus used to image molecules present in large concentrations It is used to study interactions between proteins within a cell. If two proteins are tagged with two fluorophores the merged image would show the two proteins in two colours. If the proteins interact their colours merge in vivo.
- Electron microscope is an optical instrument that utilizes electrons as a source of illumination for observing objects at a great magnification.
- When the atoms of a metal are excited by sufficient energy in the form of heat, the electron velocity is accelerated to a rate at which they leave their orbit, fly off into space and are lost to the atom. Atom bound electrons, excited by a current of high voltage, fly off from the surface of metal in a continuous stream. The electrons emitted from electron gun are passed through a collimating aperture to form a well defined beam.
- Electrons are readily absorbed and scattered by different forms of matter. Therefore the EM instrument is maintained in high vacuum. Electrons interact with atoms of specimen to form image. This interaction leads to production of many different types of rays. Transmitted electron of the beam pass straight through the specimen on to the fluorescent screen. Elastically scattered electrons interact with specimen and are scattered without any loss of energy. They deviate widely from their original path. Some electrons of the beam lose a bit of their energy while passing through the specimen and get deflected a little from their original axis of the beam. They for inelastically scattered electrons. Some electrons get back-scattered instead of passing through the specimen. Some electrons get absorbed by the specimen which then loses some of its own electrons which are emitted as secondary electrons. The electron beam might also bring configurational rearrangement in the specimen atoms that leads to production of visible light or X-rays.
- Electron Gun: The source of electrons is located on the top of the microscope column. Consists of a tungsten filament which emits electrons. The tungsten filament is surrounded by a negatively biased shield with an aperture through which the electrons are emitted. Microscopic Column: Consists of an evacuated metal tube. Electromagnetic lens of coils: Each coil is a wire wound around a hollow metal cylinder designed in such a way that when the current passes it generates a magnetic field aligned with the electron beam. Depending on the strength of the magnetic field the electron beam is deflected to various angles. Transformer Vacuum Pumps Fluorescent Screen: Captures the image generated.The screen is coated with chemicals which fluoresce upon excitation by electron beam. Water cooling system.
- Transmission electron microscopy (TEM) is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera. TEMs are capable of imaging at a significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons. This enables the instrument's user to examine fine detail—even as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. TEMs find application in cancer research, virology, materials science as well as pollution, nanotechnology, and semiconductor research. At smaller magnifications TEM image contrast is due to absorption of electrons in the material, due to the thickness and composition of the material. At higher magnifications complex wave interactions modulate the intensity of the image, requiring expert analysis of observed images. Alternate modes of use allow for the TEM to observe modulations in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging. The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first commercial TEM in 1939.
- The types of signals produced by a SEM include secondary electrons, back-scattered electrons (BSE), characteristic X-rays, light (cathodoluminescence), specimen current and transmitted electrons. Secondary electron detectors are common in all SEMs, but it is rare that a single machine would have detectors for all possible signals. The signals result from interactions of the electron beam with atoms at or near the surface of the sample. In the most common or standard detection mode, secondary electron imaging or SEI, the SEM can produce very high-resolution images of a sample surface, revealing details less than 1 nm in size. Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample.
- Ultramicrotomes used for EM samples fitted with diamond knives and microscopes. Copper grid works as the slide in EM Copper grid is placed under the floating section and then brought up. This ensuresthat the sections lay flat and uniformly on grid.
- Fracture usually occurs along planes of weaknesses. In case of biological sample, weak points are intermembrane spaces.