Re: TIRF question

Posted by Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-question-tp7584059p7584060.html

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Martin,
I would think that the refractive index of the bead is similar to glass and therefore you don't have TIRF conditions locally where the bead touches the   coverslip. But this would also imply that the beads are very close together in the aggregate and  pipe the light efficiently. Check what material they are, polystyrene has a refractive index of 1.6 at 500 nm, so this might lead to the effect. I usually see only beads on the coverslip, but sometimes very bright features of cells are visible because they are still excited in the exponential decaying field.
Best wishes
Andreas


-----Original Message-----
From: "Martin Wessendorf" <[hidden email]>
Sent: ‎01/‎08/‎2015 17:08
To: "[hidden email]" <[hidden email]>
Subject: TIRF question

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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

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