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Martin,
Some of this could also be scattered light from the TIRF laser, "piping" in
along the beads...
Independent of other optical performance, the degree of flatness of the
dichroic surface influences performance in TIRF, such that "flatter"
dichroics largely eliminate some artifacts like interference patterns in the
image or specular reflections or detection of other stray light. The level
of surface flatness that we've arrived at to be sufficiently flat (in most
cases) for a dichroic surface (measured after mounting into a microscope
filter cube) is approx: =< 0.5 waves/inch Peak-to-Valley RWD (Reflected
Wavefront Distortion) for 2mm-thick dichroics which we use as the "standard"
TIRF configuration. We also offer =< 0.25 Peak-to-Valley waves/inch
Peak-to-Valley RWD
We've found that this alone (changing to a cube in the microscope
fluorescence turret which housed a flatter dichroic)) results in better
signal/noise, and have some data indicating that this is at least partly
because fewer TIRF laser photons are propagated directly into the sample and
are instead properly reflected - maybe because of an improved surface
flatness profile. We also have much anecdotal data and customer feedback
which is consistent with greater signal/noise and lack of problematic
artifacts, and limiting of fluorescence to the TIRF zone.
This is also at least partially due to the increased levels of TIRF laser
attenuation that our fully-assembled TIRF cubes provide. At the risk of
being crass, an example albeit commercial, is described below of improving
the TIRF performance of an advanced and appropriately configured microscope
in TIRF mode detecting fluorescent beads, as you describe. In this case,
the beads are coating the surface of a coverglass and also suspended above
those beads, diluted in a layer of agarose on the surface of the coverglass.
https://www.chroma.com/sites/default/files/TIRF.pdf
With one dichroic (good quality, sputtered coating, fused silica, basic
laser quality flatness), beads were visible several microns into the sample
in TIRF mode in a 3D data set. When we simply changed cubes,
reset/reconfirmed TIRF beyond the critical angle as before, these deep
objects were not detected in an otherwise identical data set. SIM data
shows that the fluorescent objects in the first (beyond the TIRF zone) case
correlate to real objects suspended in the agarose, and undetected in the
second case. Neither changing cubes back and forth, nor any particular
order of imaging had any detectable independent effect.
Best,
Jeff
Jeff Carmichael
Technical and Product Marketing Mgr.
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Chroma Technology Corp.
an employee owned company
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Bellows Falls, VT 05101
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Andreas Bruckbauer
Sent: Saturday, August 01, 2015 2:19 PM
To: [log in to unmask]
Subject: Re: TIRF question
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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" <[log in to unmask]>
Sent: 01/08/2015 17:08
To: "[log in to unmask]" <[log in to unmask]>
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
--
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]
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