ANAT 262 Lecture Notes - Lecture 17: Confocal Microscopy, Fluorescence Microscope, Photobleaching
30 Aug 2016
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Confocal microscopy: see one focus, one slice w/o physically cutting see individual cells & inside. Can assemble images of diff sections w/computer 3d. Directed to specific point in sample, only looks at one point at any one time, scans whole sample to form image. Contains 2 controllable mirrors to direct light source. Gfp tagged proteins scan area w/high intensity light bleach area proteins in that are permanently non-fluorescent. See if other gfp tagged molecules move into that area by diffusion or if immobile. Mt 25nm in diameter, can localize as long as by itself. If come within few hundred nm difficult to resolve. Cells have fluorescent molecules background fluorescence, but not as bright as tagged proteins; still problem. Can localize as long as not near another dynein. In cell, million molecules no hope of resolving if all glowing simultaneously. If label only tiny fraction of molecules can build image at much higher resolution than lm.
