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Carl -
You are right on the mark. What I usually try to do in such circumstances
is excite just one fluor at a time and collect the emissions sequentially.
Do it this way, and the entire emission signal from each laser line can be
dumped to a single detector. Pick a broad BP filter that lets you see
whatever chunk of the emission you choose. In fact, you could use band
blocking filters to exclude just the reflected laser light. This method
won't work well with 2p excitation.
Every method has a downfall, and the downfall here is that in order to keep
from exciting the 2nd fluor (and 3rd and 4th, etc.) and producing emission
you can't isolate, you may have to excite each fluor suboptimally. Check
the absorption curves and choose an off-peak location where the other
fluors show the least absorption. Of course, this also means you need lots
of laser lines....
Sequential excitation is generally a much easier proposition if you are
able to choose your fluorophores from the very large and growing list of
direct stains (ie: DAPI) and conjugatable dyes (FITC, Alexa, Cy, etc).
Fluorescent proteins are a wrench in the works. In my experience, in any
one cell/tissue expressing two FPs, if the FPs are wavelength-neighbors
(eg: cyan and green) you are going to get overlapping emission signals.
Even with the sequential excitation technique it's better to choose FP
pairs somewhat distant in excitation wavelength (eg: cyan and yellow). When
you start trying to cram 3 FPs or more into a cell/tissue it gets Really
Messy.
Have fun,
Jim
At 05:22 PM 2/18/03 -0700, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hello all,
>In the current discussion about separating signals using narrow bandpass
>filters, I am curious about how well this actually works.
>
>If you can't separate dyes by spectral analysis, then you must have what I
>have, dichroics and barrier filters that send certain wavelengths to one
>detector and allow other wavelengths to pass to another detector. As we all
>know, the barrier (emission) filter limits what gets to the specific
>detector that the filter is in the light path for. In the scenario I
>envision, a 565 dichroic reflects light shorter than 565nm to, say, a 515/30
>bp filter which is used to allow only green light to get to that detector.
>Anything longer than 565nm gets transmitted to another llight path and
>another detector. The problem with this is that GFP, FITC or pretty much
>any other green fluorescent dye has a rather long emission tail that sneaks
>into the red spectrum. While this red light will be blocked from reaching
>detector 1 by the 515.30 bp filter, it can get transmitted to detector 2
>through the dichrioc mirror. As a consequence, narrowing the bandpass in
>detector 1 has no bearing on the bleed-through into the other detector.
>This seems particularly problematic when the emission spectra of two dyes
>overlap as much as CFP and GFP.
>
>I'm basing all of the above on the spectral analysis website:
>http://www.mcb.arizona.edu/IPC/spectra_page.htm, and after pondering why
>there can be so much bleed through from the green to red channels on our
>Nikon PCM 2000.
>Am I missing something in this analysis?
>
>Cheers,
>Carl
>
>
>Carl A. Boswell
>Dept. of Mol. Cell. Biology
>Univ. of Arizona
>(520) 626-8469
>FAX (520) 621-3709
>[log in to unmask]
>----- Original Message -----
>From: "Holly Aaron" <[log in to unmask]>
>To: <[log in to unmask]>
>Sent: Tuesday, February 18, 2003 4:47 PM
>Subject: Re: Triple labellings
>
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear Hugo -
> >
> > This should be doable. If you have a Zeiss 510-META, you should be able
>to
> > separate out the spectra. If you do not, I would suggest getting some
>very
> > narrow emission bandpass filters. This will help for either 1p or 2p
> > excitation. The 2p excitation maxima for your three dyes are quite a bit
> > apart, but there is always the possibility of overlap. CFP is max around
> > 800nm, eGFP at 920nm (some dispute over this), and dsRed out past 950nm.
>If
> > you are tuning b/t dyes, and using fairly narrow bandpass filters, you
> > should be able to separate the signals.
> >
> > Best of luck,
> > Holly
> >
> > Holly L. Aaron
> > CRL Molecular Imaging Center
> > http://imaging.berkeley.edu
> >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:[log in to unmask]]On
> > > Behalf Of Jacqui Ross
> > > Sent: Tuesday, February 18, 2003 3:22 PM
> > > To: [log in to unmask]
> > > Subject: Re: Triple labellings
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Hi Rosemary,
> > >
> > > Just interested in your comments regarding CFP excitation and
> > > emission ranges.
> > > What emission range did you collect for CFP when you were using 458nm
> > > excitation, something like 465-480nm? We found that we couldn't use
>458nm
> > > excitation for CFP and still manage to separate it out from GFP
> > > but then as I
> > > said, our signal for CFP was not the best whereas the GFP was
> > > quite bright.
> > >
> > > Cheers,
> > >
> > > Jacqui.
> > >
> > > Rosemary White wrote:
> > >
> > > > Search the CONFOCAL archive at
> > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Dear Hugo,
> > > >
> > > > As long as your CFP is bright enough so that you can collect emission
> > > > between CFP excitation, ideally ~440nm - I guess you can get this with
> > > > multiphoton(?), and GFP excitation at 488, and then collect GFP
>emission
> > > > from 500-525, say, then you should be able to separate them.
> > > We've looked
> > > > at a mixture of bacteria expressing either CFP, GFP or YFP and
> > > can separate
> > > > the three on a Leica SP2 with Ar and HeNe lasers, where
> > > excitation for CFP
> > > > is not ideal at 458nm, but where you can select the emission range.
>No
> > > > experience with DsRed, but looks as though it shouldn't be a problem.
> > > > cheers,
> > > > Rosemary
> > > >
> > > > >I was wondering if someone out there has attempted triple
> > > labellings with
> > > > >eCFP, eGFP and DsRed. Experience on double labellings
> > > eGFP/eCFP would also
> > > > >interest me. I am just a little worried the 2 might interfere. I used
>a
> > > > >zeiss multiphoton...
> > > > >I hope to hear suggestions (changing my labels is not an option... )
> > > > >Thanks
> > > > >
> > > > >Hugo Caldas
> > > > >Columbus Children's Hospital
> > > > >Columbus, OH
> > > >
> > > > Dr Rosemary White [log in to unmask]
> > > > Microscopy Centre fax 61- 2 6246 5000
> > > > CSIRO Plant Industry ph. 61- 2 6246 5475 or
> > > > GPO Box 1600 mob. 61- 0402 835 973
> > > > Canberra, ACT 2601, Australia
> > >
> > > Jacqueline Ross
> > > Biomedical Imaging Research Unit
> > > Division of Anatomy with Radiology
> > > Faculty of Medical & Health Sciences
> > > The University of Auckland
> > > Private Bag 92019
> > > Auckland, NEW ZEALAND
> > >
> > > Tel: 64 9 373 7599 Ext 87438
> > > Fax: 64 9 373 7484
> > >
> > > http://www.health.auckland.ac.nz/biru/
> > >
> >
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