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Single molecule optically viewing?

Victor De Las Casas decasas at ix.netcom.com
Sat May 16 18:23:48 EST 1998


Eyal Peleg wrote in message <01bd810a$834c1760$6b1ce8cf at default>...
>Hello
>Can anyone tell me how to view a single fluorescent molecule by an OPTICAL
>microscope.

Hello Eyal,

The only method that I am aware of for visualizing a single molecule through
an optical microscope is called Total Internal Reflectance Fluorescence
microscopy, or TIRF for short.  In this method, fluorescence illumination is
shone onto a coverslipped specimen at a critical angle, and the illumination
path is reflected by the coverslip, medium and slide to illuminate a
fluorescently labelled molecule.  This is accomplished either by the use of
external lasers at the appropriate angle, or by using a 1.2NA or greater
long working distance objective, and placing a dark stop covering the
central area of the illumination path (to prevent illumination from hitting
the specimen which is not at the proper angle).  In essence, you're using
reflected light darkfield fluorescence microscopy.

>What is the technical information of his microscope (objective, light
>source)

At the present time, there are very few microscope reps who even know what
this technique is, much less how to configure a scope to allow for this type
of technique.  Depending on your area, you may want to contact your local
rep and see.  There are a couple of companies working on objectives which
make this a simple technique, but no one has a turnkey solution yet.  Look
toward Leica as the leader in innovation in this area.


>Are there any instructions for the specimen preparations?
>(fluorescent dye etc.).


I'd suggest doing a yahoo search for TIRF, and seeing what you dig up.  This
research is going on at some of the larger and more prestigious Universities
and research centers in the US, including UCLA, University of Pennsylvania,
and NIH.


Hope this helps,


Victor





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