Fluorescence and electron microscopy

Principle and basic instrumentation of advanced microscopes like fluorescence, confocal microscopes and Electron microscopes

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, Mumbai, INDIA

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

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

8. Why does Stoke’s shift occur? Energy levels Ground state (no light absorbed) Excited state (light energy absorbed ) Each energy level is divided into Vibrational Energy level Rotational Energy Level 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

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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

13. Instrumentation Light Source : Xenon Lamp or Mercury Arc Lamp. Should provide UV and visible light. Excitation Filter: Selects the wavelength of light absorbed by fluorophore . Dichroic Mirror: Reflects the light coming from light source and transmits the light coming from specimen. Lens System : objective and ocular lens. 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

14. Applications Non-specific dye binding Immunofluorescence GFP-tagging created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

19. Limitations of Fluorescence Microscopy Fluorophore used might interfere with metabolic pathway studied. E.g. GFP is a large protein and might affect movement of tagged protein. Excitation light might damage live tissue Excited fluorophore might react with oxygen and generate free radicals toxic to cell. 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

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

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, Mumbai, INDIA

22. Instrumentation Light Source – Zirconium Arc Lamp or Laser Light Source 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. Objective Lens- focuses the fluorescent light from each point on specimen to detector pin-hole Dichroic Mirror Pin-hole – Eliminates out-of-focus light. Two types – Light source pin-hole and Detector Pin-hole Detector – Photomultiplier tube created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

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, Mumbai, INDIA

24. 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

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, Mumbai, INDIA

26. 3D image made by Z-stacking created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

27. Applications Imaging highly expressed molecules. Protein interactions within the cell To study 3D architecture of cells 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

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, Mumbai, INDIA

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 Condensor 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

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

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, Mumbai, INDIA

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, Mumbai, INDIA

33. Instrumentation Electron Gun: Located on the top of the microscope. Its is a tungsten filament in a negatively biased shield with an aperture. Microscope Column: Evacuated metal tube. All components aligned one on top of the other. Provides shielding from X-rays. 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. Transformers: provide high voltage current to the electron gun. Vacuum Pump : Maintain vacuum within the microscope column. Fluorescent Screen: for image capture Water Cooling system: Prevents over-heating. created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

34. Working Image Formation Magnification Resolving Power created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

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, Mumbai, INDIA

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 1000000X created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

37. Resolving Power Limited by wavelength of illuminating electron bean. Limit of resolution = half the wavelength If λ = 0.037 A o then D (limit of resolution) = 0.018 A o Practically, best resolution achieved is 4 – 10 A o created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

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, Mumbai, INDIA

42. Shadow Casting A technique used to improve contrast. Sample on copper grid is placed in evacuated chamber Heavy metal like chromium, palladium, platinum or uranium is evaporated at an angle from a filament 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. 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

43. Negative Staining Involves treatment of material with phosphotungstic acid The stain penetrates the empty spaces of the cell 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

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, Mumbai, INDIA

45. Freeze Fracture - TEM Done to impart a 3D texture and better resolution to image. Specimen tissue frozen at -130 degrees in liquid freon Specimen is transferred to an evacuated chamber at -100 degrees Microtome used for cracking or fracturing tissue. Fractured sample is left in vacuum or removed in water Specimen is then subjected to shadow casting created by: Ms. Shmilona Jain, Assistant PRofessor, Biotechnology Department, VES College of Arts, Science and Commerce, Mumbai, INDIA