Microscopy Flashcards
freeze slammer limitations
freezing depth - tissue must be thinnner than 10 microns
electric stimulation requires wires, and only excitable cells can be used
array tomography
cut serial sections of fixed tissue on ultramicrotome, image immunostained sections separately, then computationally collate and align section images
electron microscopy anatomy
electron gun, condensor magnets, viewing screen or photographic film
structured illumination microscopy
Exciting light filtered with a variety of illumination patterns, then image is reconstructed using interference of exciting light with sample structure. Produces a much crisper image
PALM
photoactivated localization microscopy, one type of superresolution method
confocal fluorescence microscopy
fluorescent specimen illuminated with a focused point of light from a pinhole, only emitted fluorescent light from in-focus point reaches detector. Out of focus point emissions largely excluded
resolution of conventional light microscopy
200 nm
Which type of microscopy is best for thick specimens?
two photon, or array tomography for very thick tissues
which microscopy is best for cell surface imaging?
TIRF
why use FRAP?
measures molecular diffusion in cells
limits of superresolution atm?
10 nm
how do you prepare samples for EM?
fixing, staining, sectioning or heavy metal shadowing
resolution of cryo EM
10-20A structures
autofluorescence
when cells and tissues emit light with native weak chromophores - is common, and thus makes it a requirement that a parallel control sample lacking the specific fluorophore be included
photobleaching
happens with destruction of the fluorophore, can be counteracted with additives sometimes but usually most effective to minimize exposure to excitation beam when capturing images
