Dear Shiva, there are so many possible combinations of stains and
     fluorescent probes that in the end, you have to use logic. The
     excitation and emission spectra of a large number of fluorescent
     dyes can be accessed from either fluorescence.bio-rad.com (which
     also allows superimposition of spectra with different emission
     filters) or www.probes.com. If not, just use the Bio-Rad
     fluorophore wall chart to read off the peak excitation and emission
     wavelengths. Obviously, you want to collect as much light as
     possible from each dye. If the emission spectra of your dyes do not
     significantly overlap, you should be able to perform simultaneous
     collection in a multi-labelled sample. If the spectra overlap a
     lot, you should perform sequential collection to avoid fluorescence
     bleed-through. Your confocal system should have dichroic mirrors
     which will either be long pass (LP) or short pass (SP). If for
     example, you have a green emitting dye and a red emitting dye in
     the same sample, you need to use a dichroic which will split the
     green signal from the red signal. Typically, this would be
     something like a 560LP which will reflect the shorter green
     wavelengths into one channel and 'pass' the longer red wavelengths
     into the second channel. To be more selective about the range of
     wavelengths you collect into your detectors, you can then use the
     most appropriate emission filter for the dye you are using. Many
     confocal systems will automatically set the collection parameters
     for a particualr type of dye combination. All you need to do is to
     open this as a Method from your software.

     I hope this helps

     Best regards

     Anna Smallcombe PhD
     Senior Applications Specialist
     Bio-Rad Microscopy Division


______________________________ Reply Separator _________________________________
Subject: combination list of stains with
Author:  Confocal Microscopy List <[log in to unmask]> at
Internet
Date:    02-10-2000 5:21 pm


Hi Confocalists
Does any one have a table/list which gives the details as to which beam path
and channel assignment should be used for different stains/ stain
combinations? If not, could someone tell me how to figure out these
combinations.

Regards, Shiva
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