Microscopes and Microscopy

1. Microscopes and Microscopy MCB 380 Good information sources: Alberts-Molecular Biology of the Cell http://micro.magnet.fsu.edu/primer/ http://www.microscopyu.com/

2. Approaches to Problems in Cell Biology • Biochemistry-You can define a enzyme reaction and then try to figure what does it, when, where and under what control • Genetics- You can make a mutation and then try to figure out what you mutated • Cell Biology- You can visualize a process and try to understand it- for instance cell division was one of the earliest • Today- there are no distinctions. You cannot be just one thing, or be knowledgable about one thing. You need to take integrated appoaches to problems using the appropriate tools when needed. If you limit your approach, you limit your science

3. Properties of Light • Reflection • Diffraction-scattering of light around edges of objects • Limits the resolution • Refraction- bending of light when changing medium (index of refraction) • principle that lenses use to focus light • Used in contrasting techniques • Interference • light waves can subtract and add • Polarization- allowing only light of a particular vibrational plane

4. Refraction Diffraction

5. Interference Constructive Destructive

6. Limitations • light waves diffract at edges-smearing causes limits • resolution = minimum separation of two objects so that they can both be seen

11. Limit of Resolution • The cone of light collected by the lens determines the resolution (nsinq) n=refractive index • Max NA is 1.4 (refractive index of oil) • Lenses range from 0.4-1.4 NA • Maximum magnification is about 1000x Resolution = 0.61l/nsinq = 0.61l/NA

15. Resolution of Microscopes • Visible light is 400-700nm • Dry lens(0.5NA), green(530nm light)=0.65µm=650nm • for oil lens (1.4NA) UV light (300nm) = 0.13µm • for electron microscope • l=0.005nm but NA 0.01 so =30-50nm

16. Sizes of Objects • Eukaryotic cell- 20µm • Procaryotic cell-1-2µm • nucleus of cell-3-5µm • mitochondria/chloroplast- 1-2µm • ribosome- 20-30nm • protein- 2-100nm

17. Microscope Objectives • complex combinations of lenses to achieve • high magnification • low optical distortion • Low chromatic distortion • flat field

18. Contrast • Cells are essentially water and so are transparent • In addition to resolution and brightness, you need to generate contrast to see things • Two objects may be resolvable by the microscope, but if they don’t differ from the background, you cannot see them • Contrast can be accomplished with staining or optical techniques

19. Microscope types • Brightfield • Stereo • Phase contrast • Differential Interference Contrast • Fluorescence • Confocal • Electron • Transmission • Scanning • Atomic Force

20. Microscopes • Stereo • Different images are sent to the two eyes from different angles so that a stereo effect is acheived. This gives depth to 3D objects • Brightfield • use a prism to send the light to both eyes • light passing through specimen is diffracted and absorbed to make image • Staining is often necessary because very low contrast

22. Phase Contrast • A phase ring in condenser allows a cylinder of light through in phase. Light that is unaltered hits the phase ring in the lens and is excluded. Light that is slightly altered by passing through different refractive index is allowed through. • Light passing through cellular structures such as chromosomes or mitochondria is retarded because they have a higher refractive index than the surrounding medium. Elements of lower refractive index advance the wave. Much of the backround light is removed and light that constructively or destructively interfered is let through with enhanced contrast • Visualizes differences in refractive index of different parts of a specimen relative to the unaltered light

23. Hemotoxylin Stain of Chromosomes (18.5)

24. Phase Contrast

25. Differential Interference Contrast or DIC or Nomarski • A prism is used to split light into two slightly diverging beams that then pass through the specimen. • On recombining the two beams, if they pass through difference in refractive index then one retarded or advanced relative to the other and so they can interfere. • By changing the prism you can change the beam separation which can alter the contrast. • Also measures refractive index changes, but for narrowly separated regions of light paths-ie it measures the gradient of RI across the specimen • Gives a shadowed 3D effect • Optically sections through a specimen

26. DIC beam Path

29. Interference Reflection Microscopy • Looks at light reflected off the surface only. • By polarizing the light and then analyzing the resultant, can see differences in height of reflecting surface. • If something is closely opposed to the glass surface, then it does not pass through a new medium and when reflected back it is eliminated. • Altered light is left in and looks light while closely apposed is dark.

30. IRM Light Path

31. IRM Images

32. Total Internal Reflection Microscopy • Light shined on a reflective surface at an appropriate angle will generate an evanescent wave, a wave of energy propagating perpendicular to the surface • It only propagates about 100-200nm from the surface • Allows one to visualize events taking place near the membrane (exocytosis, cytoskeleton)

33. Evanescent Wave http://www.olympusmicro.com/primer

35. Specimens • Live cells or tissue- • can you see the structure in a live cell? • can you image the cell without damaging it with light? • Fixed-try to retain structure intact • Glutaraldehyde- reacts with amines and cross links them-destroys 3D structure of many proteins • Formaldehyde-reacts with amines and cross links them • slower reaction, reversible, not as extensive • Methanol, acetone, ethanol, isopropanol- precipitate material- not as good for retaining structure • Rapid freeze (liquid helium)- then fix

36. Fluorescence Microscopy

37. Fluorescence Microscopy • Fluorescent dye- a molecule that absorbs light of one wavelength and then re-emits it at a longer wavelength • Can be used alone or in combination with another molecule to gain specificity (antibodies)

38. Epifluorescence Microscope

39. Dead cells stained with a Fluorescent reagent (fluorescent phalloidin- a fungal toxin) to visualize actin filaments

40. Endoplasmic Reticulum Stained with a synthetic dye that dissolves in ER membranes

41. Brightness of an image

42. Discussion Problem • Actin filaments are 8nm in diameter • We can see a single filament with phalloidin stain in fluorescence microscope • The resolution limit of the microscope is 200nm • WHY CAN WE VISUALIZE THE FILAMENT??

43. Co-localization of Proteins • FRET- Fluorescence Resonance Energy Transfer • If the emission wevelength of one probe overlaps with the excitation wavelength of another probe you can get resonance energy transfer • Non-radiative transfer- the energy is transferred directly from molecule to molecule • The two molecules need to be within 10 nm because the energy transfer falls off with the 6th power of distance • You excite with the donor wavelength and measure emission at the recipient wavelength

44. Monitor interactions between two proteins. Left: CFP-NIPP1, center: YFP-PP1, right: FRET. Top: Both YFP-PP1g are expressed. NIPP1 binds and retargets PP1 to nuclear speckles outside of nucleolus. Bottom: Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget speckles from nucleolus. After bleed-through correction, minimal FRET can be observed (right). Images acquired during 2002 FISH Course CSHL Labs (Universal Imaging Website).   

45. Co-localization of proteins • FLIM-Fluorescence Lifetime Imaging • When a probe is excited briefly, the rate of decay of fluorescence is different for each probe-so if you have different probes in the cell you can characterize them based upon lifetime • FRET-FLIM- measure the decay of the donor during FRET

46. Confocal Microscopy • Fluorescence microscope • Uses “confocality” (a pinhole) to eliminate fluorescence from out of focus planes • Minimum Z resolution=0.3µm • Because you can optically section through a specimen, you can determine the localization of probes in the Z dimension • You can also build 3D (4D) models of structures and cells from the data

47. Laser scanning confocal • Uses a laser to get a high energy point source of light • The beam is scanned across the specimen point by point and the fluorescence measured at each point • The result is displayed on a computer screen (quantitative data)

48. Laser scanning confocal Microscope http://www.microscopyu.com

49. Specimen illumination

50. Results