1. Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)

7. Resolution of biological objects

8. Capturing and interpreting light images

9. Different methods to visualize cellular morphology and objects

10. Light and fluorescence microscopy

11. Detection methods for subcellular structures

12. Methods to better resolve objects in 3D

19. Relative sizes On the microscopic to Macroscopic scale

20. Cellular proteins can be visualized in Real time in living cells S. CEREVISCIAE - BAKERS YEAST GOLGI GREEN AND RED FLUORESCENT PROTEINS

21. How Flourescence works

22. A.VICTORIA (AKA - GLOWING JELLYFISH) Isolate protein - green fluorescent protein (GFP) Abs. Max = 488nm Now can fuse protein of interest with GFP GFP Getmeoutaherenow 5’ 3’ UTR AUG UAG UAA UGA DNA plasmid GFP Getmeoutaherenow Making Fluorescent Cells

23. Ta da…..The Brainbow Making Fluorescent Cells GFP is a Beta-can  -helices  -sheets chromophore Cerulean GFP Banana Orange…….

24. d = 0.61   n sin  Resolving power D = minimum distance of 2 points = wavelength n = numerical aperture  = angle of cone of light (1/2)

25. Methods to increase contrast Specific stains Light microscope comparisons Bright field Phase contrast Nomarski CFP YFP axons

26. Out of focus light can be removed by computers Raw Deconvolved

27. Other uses of Fluorescent proteins FRET - fluorescence resonance energy transfer -can detect changes in interactions -donor FL energy reduced while acceptor increased Bi-molecular complementation Caged proteins

28. Light microscope Electron microscope Electron microscopy 1nm resolution Negative stain Shadow casting

29. Sample preparation is time consuming

31. Onion root cell Freeze-fractured Ciliary axoneme - deep etch Insect head - SEM - bacteriophage