1. Molecular Cell Biology Light Microscopy in Cell Biology Cooper Modified from a 2010 lecture by Richard McIntosh, University of Colorado

2. Images from a light microscope can be strikingly informative about cells How are these images made? What questions can they answer? What are their limitations? Can you make and use them?

3. Scales of absolute size: powers of 10

4. Wavelength sets limits on what one can see Light behaves as a Wave

5. Resolution = 0.61 x wavelength of light NA (numerical aperture) The effect of NA on the image of a point. The need for separation to allow resolution θ θ θ Lower limits on spatial resolution are defined by the Rayleigh Criterion NA = nsinθ n = refractive index of the medium θ = semi-angle of an objective lens

6. Contrast in the Image is Necessary: Types of Optical Microscopy Generate Contrast in Different Ways Bright field - a conventional light microscope DIC (Differential Interference Contrast - Nomarski) Phase contrast Fluorescence Polarization Dark field

7. Bright-field Optics: Light Passing Straight Through the Sample Most living cells are optically clear, so stains are essential to get bright field contrast Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable Fixing and staining is an art

8. Classic drawings and modern images made from Giemsa-stained blood smears Plasmodium falciparum Histidine-rich Protein-2

9. Generating Contrast Staining Coefficients of absorption among different materials differ by >10,000, so contrast can be big Without staining Everything is bright Most biological macromolecules do not absorb visible light Contrast depends on small differences between big numbers Need an optical trick

10. Mammalian Cell: Bright-field and Phase-contrast Optics

11. Principles of bright field and phase contrast optics

12. Differential Interference Contrast (DIC) Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen Small amounts of contrast can be expanded electronically Lots of light: Video camera with low brightness & high gain

13. Brightfield vs DIC

14. DIC has shallow depth-of-field: Image a single plane in a large object Worm embryo

15. DIC: Good contrast. Detection vs Resolution. Microtubules: 25 nm diameter (1/10 res.lim.) but visible in DIC

16. Fluorescent staining: High signal-to-noise ratio (white on black)

17. Principle of Fluorescence Absorption of high-energy (low wavelength) photon Loss of electronic energy (vibration) Emission of lower-energy (higher wavelength) photon

18. Design of a Fluorescence Microscope

19. Fluorescent tubulin injected into a Drosophila embryo, plus a DNA stain

20. Green Fluorescent Protein - Considerations Color - Not just green Brightness Time for folding Time to bleaching

21. Live-cell Imaging of Microtubule Ends: EB1-GFP chimera

22. GFP-Cadherin in cultured epithelial cells

23. Immunofluorescence Primary Abs recognize the antigen (Ag) Secondary Abs recognize the primary Ab Secondary Abs are labeled

24. Immunofluorescence Example Ab to tubulin Ab to kinetochore proteins DNA stain (DAPI)

25. Biological microscopy problem: Cells are 3D objects, and pictures are 2D images. Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections” In an image of one section, one must remove light from other sections Achieving a narrow “depth-of-field” A “confocal light microscope”

26. Laser-Scanning Confocal Light Microscopy Laser thru pinhole Illuminates sample with tiny spot of light Scan the spot over the sample Pinhole in front of detector: Receive only light emitted from the spot

27. Light from points that are in focus versus out of focus

28. Spinning-disk confocal microscopy: Higher speed and sensitivity

29. Example: Confocal imaging lessens blur from out-of-focus light

30. Optically Sectioning a Thick Sample: Pollen Grain Multiple optical sections assembled to form a 3D image

31. 3D Image Reconstructed From Serial Optical Sections Obtained with a Confocal Microscope

32. Fluorescence can Measure Concentration of Ca 2+ Ions in Cells: Sea Urchin egg fertilization Phase Contrast Fluorescence

33. Summary Light microscopy provides sufficient resolution to observe events that occur inside cells Since light passes though water, it can be used to look at live as well as fixed material Phase contrast and DIC optics: Good contrast Fluorescence optics: Defined molecules can be localized within cells “Vital” fluorescent stains: Watch particular molecular species in live cells

34. End