A reference to our work using TIR to see single copies of GFP at the University of California San
Diego (UCSD) is Nature 388, p. 355 (1997).
Best regards, W. E. Moerner
Prof. W. E. Moerner wmoerner at ucsd.edu
Mail Code 0340, Urey Hall 3226 Phone 619-822-0453 Fax 619-534-7244
Department of Chemistry and Biochemistry, 9500 Gilman Drive
University of California San Diego
La Jolla, CA 92093-0340 http://chem-faculty.ucsd.edu/moerner
From: Victor De Las Casas [SMTP:decasas at ix.netcom.com]
Sent: Saturday, May 16, 1998 4:24 PM
To: fluorpro at net.bio.net
Subject: Re: Single molecule optically viewing?
Eyal Peleg wrote in message <01bd810a$834c1760$6b1ce8cf at default>...
>Can anyone tell me how to view a single fluorescent molecule by an OPTICAL
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
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,
Hope this helps,