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Dear List--

I'm a newbie to TIRF microscopy and have a question. Using our Zeiss 
TIRF 'scope, I can often clearly image structures in my biological prep 
(cultured cells) that are in a deeper focal plane than the cover slip. 
This is true even using fairly high TIRF angles (e.g. 80 degrees).

Using TIRF, I would expect that only one focal plane would be visible: 
the plane in contact with the cover slip.  This mostly appears to be the 
case when I'm imaging a dilution of fluorescent beads: using epi 
illumination, I can see beads throughout the thickness of the sample 
whereas using TIRF illumination, I can only see those beads that are 
stuck to the cover slip, or that transiently diffuse close enough to the 
coverslip that they flicker into appearance for a moment or two.  
However, if clumps of beads have aggregated and are in contact with the 
cover slip, I can image these beyond the plane of the cover 
slip--sometimes a micron or more beyond.

Anyone got an explanation for how this occurs?  Light piping?  Are some 
structures just so bright that the evanescent wave is able to excite 
them, even at that distance?

Thanks!

Martin Wessendorf

-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
Minneapolis, MN  55455                    e-mail: [log in to unmask]