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Hey, George--

On 6/23/2013 9:56 AM, George McNamara wrote:

> I disagree with a couple of the authors imaging advice on page 544 (most
> of the advice is very good):
>
> Authors: "Fluorophores with overlap in the excitation or emission
> spectra should be imaged sequentially rather than simultaneously to
> minimize fluorescence cross-talk and thereby optimize color separation".
>
> On a typical confocal microscope (ex. Leica SP5, SP8 or Zeiss LSM710,
> 780) there are several detectors (ex. 5 on SP5 or SP8) or detector array
> (ex. LSM710 or 780). Therefore, fluorophores that excite well at a given
> excitation wavelength should be imaged simultaneously. I also recommend
> the latest detectors, ex., HyD for leica, GaAsP for Zeiss, photon
> counting mode if available (and TCSPC lifetime if available).

I'm confused about why you think this is bad advice.  If you have 
coexpression of labels, it'll be much easier to determine whether or not 
a particular cell is single-labeled or multiple-labeled if you use a 
laser that doesn't excite all the candidates (or conversely, if you use 
a barrier filter that doesn't pass all the candidates).  This is 
particularly true when you have strong expression of one and weak 
expression of the other.  Even with an detector array, there's a limit 
to what you can ferret out (--unless you have infinite photons, of course!)

However, my confusion probably means I'm missing something so explain away!

Thanks--take care--

Martin Wessendorf


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