This is quite interesting, since Ian Harper and I were just discussing
the issue of confocally-detected Trypan Blue fluorescence in some
detail. I'm forwarding our discussion (assume you don't mind, Ian!) to
see if anyone else can make sense of what's happening.
Mark Rand, Ph.D.
U.W. Dept. Neurology, School of Medicine
1959 NE Pacific St., Box 356465
Seattle WA 98195
fax: (206) 616-7211
vox: (206) 616-5153
Forwarded discussion follows:
On Fri, 14 Nov 1997 16:23:04 +1000 [log in to unmask] (Ian
Harper) wrote:
>Hi Mark
>
>I certainly remember you , as well as your puffing paint project.
>Sorry not to have replied sooner, it's grant hunting season round
>here !!
>
>> Hi Ian,
>>
>> We met at Jim Pawley's course 2 summers ago. Your message reminded
>> me of something I'd seen with a BioRAD 500 confocal but couldn't
>> replicate with a wide-field fluorescence imaging system, namely that
>> a 0.4% solution of Trypan Blue pressure-applied through a
>> micropipette gives excellent confocal images of the applied solution
>> using Argon-ion laser (488 nm) illumination, but no detectable
>> signal using a cooled CCD camera and 488 nm monochromator
>> illumination. It doesn't make sense to interpret the confocal image
>> as a reflected signal, since the BioRAD dichroic and emission filter
>> would have excluded emissions below about 514 nm (it was the
>> standard BioRAD cube for fluorescein; we were using it for fluo-3
>> imaging). I also saw this effect using a BioRAD 600 with
>> Argon/Krypton-ion laser (488 line excitation) and the same (nominal)
>> filter cube set.
>
>This doesn't sound quite right. I may be wrong, but I don't think
>Trypan Blue fluoresces significantly at all. Is there any chance that
>you used another dye, say for example Eosin ?
>
I'm certain that it was Trypan Blue. There were another strange aspect
of the fluorescence: it was much more pronounced when the
Trypan/Ringer's solution contained 10 mM caffeine. Actually, I think I
was able to get the same result using the Noran system at the confocal
course; at least, I don't remember *not* getting the expected
fluorescence signal with the puff of Trypan/caffeine that I was
administering to Tim Murphy's hippocampal neurons (well, not *his* per
se, but you know what I mean). The only guess I can make at this point
is that the intensity of the laser illumination can cause Trypan to
fluoresce at green wavelengths.
>>
>> With the wide-field fluorescence imaging system I presently use, an
>> excitation wavelength scan of Trypan Blue in Ringer's solution shows
>> a peak at 385 nm when monitoring the 520 nm emission, and when small
>> volumes of 0.4% Trypan Blue are pressure-applied via micropipette
>> into a low-volume, rapid-turnover bath perfusion chamber, the
>> fluorescence elicited by the 385 nm excitation *is* modestly
>> detectable with the cooled CCD camera using short integration times.
>> Excitation wavelength scans of the Trypan solution above 420 nm show
>> very little emitted fluorescence, so I'm still puzzled by the
>> ability of the confocal to capture the Trypan Blue signal using the
>> 488 line. One way to test the possibility that it is due to
>> reflectance would be to exchange the reflectance filter cube for the
>> fluorescein filter cube and see whether the Trypan signal increases,
>> but unfortunately I don't have access to a confocal anymore. If you
>> have any thoughts on this I'd appreciate hearing from you.
>
>Well, as you say Trypan Blue doesn't fluoresce in theB/G region. But
>what are you after in terms of your fluorescence imaging. If you need
>to use a dye much like a smoke bomb to check for current
>strength/drift etc, as I recall you were doing, just use sodium
>fluorescein.
>It's cheap, non toxic, and won't enter the cell.
This sounds like a much better alternative, particularly since the
Trypan 385 signal is only weakly detected with my current imaging
system. We're buying a rapidly-switching monochromator (less than 3 ms
between any wavelength) which should be able to capture the 345/385
ratio pair as well as the blue-induced fluorescein signal during short
stimulations, provided the long-pass value of the dichroic is higher
than the wavelength used to excite the fluorescein. I'll give it a try
and let you know how it works out.
***********************************
On Fri, 14 Nov 1997 13:10:09 -0400 [log in to unmask] (Carol Bayles)
wrote:
>Hello all,
> I was interested in L Baker's use of trypan blue to quench
>fluorescence in the FITC channel. In fact, trypan blue itself
>fluoresces
>in the red channel of our confocal microscope. I have tried in vain to
>find any specific info on excitation or emission spectra. The only
>mention of the fluorescence properties of trypan blue I found is in a
>paper
>by Krause, et al (incl Watt Webb) 1984 which contained the statement
>"No
>relationship between trypan blue fluorescence and viability was
>observed
>(results not shown)". Does anyone have any more info on trypan blue
>fluorescence? It seems to me this could be a very valuable property
>that
>has not been exploited.
> Thank you for any info.
>
>Carol Bayles
>
>
>
>>Y'all- the following question came from one of our users - any
>>help/references would be greatly appreciated.
>>
>>In FACS analysis it is possible to quench autoflourescence in the FITC
>>PMT by adding trypan blue to the FITC-labelled secondary antibody,
>>without significant quenching any of the specific staining. We are
>>having some problems with autoflourescence in lymphnode frozen
>sections
>>from mink. Does anybody know if it is possible to quench
>>autoflourescence in flourescence microscopy by trypan blue or by other
>>means???
>>Thank you in advance......
>>
>>Lu
>>
>>Lucia P. Barker, Ph.D.
>>Microscopy Branch
>>Rocky Mountain Laboratories
>>903 South 4th Street
>>Hamilton, MT 59840
>>
>>Telephone: (406) 363-9252
>>FAX: (406) 363-9371
>>Electronic Mail: [log in to unmask]
>
>
>Carol Bayles
>607-254-4860
>Flow Cytometry and Imaging Facility 607-254-4847 fax
>Biotechnology Resource Center [log in to unmask]
>160 Biotech Bldg
>Cornell University
>Ithaca NY 14850
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