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Post by anamikariya » Wed Mar 21, 2012 3:33 pm

What additional information can be obtained by using fluorescence microscopy over light microscopy ??

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Post by jonmoulton » Wed Mar 21, 2012 8:52 pm

A fluorescence microscope can reveal whether there are detectable concentrations of fluorescent material in a sample and how the material is distributed in the sample.

However, to check for different excitation wavelengths and emission colors, you must switch part of the optics in the microscope. This is easy in most fluorescence microscope as you slide a different "cube" into the light path. A fluorescence microscope cube contains a filter for the excitation light that lets "usually" a fairly narrow band of wavelengths through to reach the sample, a dichroic mirror used to split excitation and emitted light, sending them along different paths and an emission filter which helps prevent excitation light from reaching the observer's eye.

The technique is interesting and useful because there are many fluorescent probes which can be applied to a sample to reveal different structures. Sometimes they are small fluorescent molecules applied alone, similar to the stains you might be familiar with from histology or microbiology. Sometimes they are fluorescent groups linked to antibodies that will bind to some particular structure in the sample. Since these fluorescent molecules or labelled antibodies often accumulate at some cellular structure of interest, they can be valuable in showing where such structures occur in samples.

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Post by marquitosgm » Thu Mar 22, 2012 10:15 am

A very common application of fluorescent microscopy in cell biology is immunoflorescence.

It helps to reveal protein structures which are recultant to show themselves by usual, more simple, optic microscopy techniques. Cytoskeleton is a good example.

Briefly, proteins under study have to be isolated first from cell extracts. Then, specific antibodies are obtained and a fluorescent mark is atached to them by chemical methods. Fluorescent antibodies will selectively attach to the target protein in a sample, revealing it's location when illuminated with UV light.

Ever seen those fancy images of cell skeleton seeming a spider's web over a black backgorund? That's what you get.

Hope this helps and is not too obvious.

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