cellbiotest1 - lecture 1 resolution ability of microscope...

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lecture 1 - resolution: ability of microscope to identify two objects separated by distance ‘x’ as distinct - key limits to resolution = wave length of source of illumination - theoretical limit of resolution is about ½ wavelength of source of illumination - for light microscopy (LM) resolution about 250 nm (½ wavelength blue light) - for EM about 0.2 nm, ‘real’ limit with most biological specimens about 2 nm - super resolution microscopy – form of light microscopy - has limit of about 10-100 nm and thus exceeds supposed limit of resolution of LM - why not use EM all the time? - cannot be used with living specimens because EM requires a vacuum - really expensive and time consuming - EM is not appropriate when your sample is large or want more ‘global’ view - if you are going to use non-living samples – preparation steps - fix sample: apply chemical that kills cell and freezes (fixes) everything in place - fixing provides one with a snapshot of what was occurring at that time - permeabilization: use non-ionic detergent to poke holes in membranes of sample - allows material penetrate sample - stain sample: soak sample in ‘stains’ to give sample contrast - bind generally to cell structures and differentially absorb or reflect life (for LM stains) - for EM stains bind generally to cell structures and absorb/reflect electrons - sectioning sample: slicing thin to allow source of illumination to penetrate sample - stains let us see general structures, however to truly understand how cells work we must know where specific macromolecules are in cells - probe: reagent that lets us follow the distribution of a specific cell constituent in cells - to generate probes: - link something that binds to cell constituent of interest to a ‘reporter’ (allows simple detection of other part of proge) - link constituent of interest itself to reporter - reporters: usually fluorescent molecules ( fluorophores) - excite by one wavelength of light and emit a different wavelength of light - ex flourescem, Alexa 488 (both excited by blue light and emit green light) Cy 5 ( excited by red, emits far red), rhodamine ( excited by green, emits red) - use fluorophores with fluorescence microscope - can illuminate samples with different wavelengths of light and can collect different wavelengths coming from sample ( SEE PICTURES IN NOTES) - use fluorescence to detect specific proteins in sample via “immunoflourescence” - immunoflourescence: use of antibodies coupled directly or indirectly to flourophores to detect specific proteins - probe will be combo of flourophore and antibody - antibody: protein that binds specifically to other proteins - to generate antibodies: purify protein and inject into animal such as rabbit, chicken, mouse – animals generate antibodies against protein - purify antibodies and experiment is ready 1. purify protein of interest using biochemistry-based approaches 2. inject purified protein into animal 3. wait several months for animal to mount immune response against inject protein
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This note was uploaded on 02/29/2012 for the course ZOOLOGY 570 taught by Professor Stretton during the Fall '10 term at University of Wisconsin.

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cellbiotest1 - lecture 1 resolution ability of microscope...

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