Fluorescence Microscope - (Apr/26/2007 )

Hai,
I have a question about fluorescence microscopes.
These are integrated with an mercury lamp.
What is the exact reason of this lamp, and why can't use a coloured filter with an normal lamp?
I want to visualize GFP bacterias, but I dont have a good fluorescence microscope.
Does anybody know what other technique I can use?

Thanks for helping!

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The main reason for using a mercury lamp is the intensity of light it produces. There are other non-mercury super-high intensity lamps out there too. Using a excitation filter, you filter most of the spectrum out in order to get a specific wavelength for excitation, in so doing you lose all of the of the intensity contributed by the sum of all of the other wavelenghts. If you were to use a standard lamp with a colored filter, you'd need super long exposures in order to get a useful image, which may in turn photobleach your fluorophore.

Check out this tutorial <http://www.microscopyu.com/articles/fluorescence/index.html> which explains this a lot more thoroughly than I. Cheers!-JAH

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You may want to check into some LCD lights on the market that give rise light with wavelength that is close enough for FITC/EGFP.

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Mercury arc lamps provide excitation wavelengths and intensity that can be used for a variety of standard fluorescence reagents.

The other consideration you need to be mindful of is the emission wavelength of the fluorophore you are using. A good fluorescence microscope will have a filter that is capable of separating the illumination and emission wavelengths so that all you see through the objective is the green emission wavelengths and not all the background blue excitation light (in the case of GFP). This is fundamentally different than transmitted light microscopy (example phase contrast or DIC) in which the image is generated based on differences in the light path through the sample.

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