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Giovanna Borsellino wrote:

> I am at the beginning of my "transition" from flow cytometry to confocal 
> imaging (and I'm loving it!), and I have some basic questions.
> We have a Zeiss LSM510 equipped with  488, 546, and 633 lasers.
> I would like to use Qdots to avoid this horrendous bleaching problem. 
> The questions are:
> 1. Can I use Qdots 700, or will the signal be too low (low energy far red)?

My guess is that the 705 nm dots would work, though I think that the 655 
nm dots (or Cy5) would produce more of a kick at the PMT.  --For 
whatever it's worth, I notice that the quantum dot company show two 
images of labeling obtained using the 655 nm dots, but none from the 705 
nm dots: http://www.qdots.com/live/render/content.asp?id=95 .

> 2. Can I use three different Qdots on the same sample and collect them 
> from the same "line"? How about two? If their emission peaks are indeed 
> so tight, maybe it will be possible?

This is a complicated question and the answer depends in part upon 
whether or not a given "point" in your sample is labeled by more than 
one fluorophore.  In principle it's possible and may be worth doing when 
the fluorophores are not in the same structure.  In practice, it may be 
best to excite each fluorophore selectively, at least until you're 
certain of the characteristics of your labeling.

I think that there may be a fundamental difference between the ways that 
confocal microscopy and FACS are used; understanding this difference may 
make it easier to understand why strategies that are fine in 
cell-sorting might not always be the best in microscopy.  If I 
understand correctly, in cell sorting, you ask questions of populations 
of cells--i.e. by examining large numbers of cells, you can average out 
the noise.  In microscopy, you often ask questions of particular cells. 
  Thus signal-to-noise needs to be higher.

> 3. Why don't people ever use APC (allophycocyanin) in microscopy, while 
> we use it all the time in flow? The bleaching I see is comparable to 
> that of Cy5, at least to my very unexperienced eye.

I tried it back about 15 years ago and had no luck, so I dropped it and 
went to Cy5 when that came out.  (I found out afterwards that I'd ruined 
the APC by running it through a freeze-thaw cycle.)  So why don't I use 
APC?  Habit.  It works well and I like the fact that I can mount slides 
with permanent mounting medium (DPX), throw the slides in a box, and 
look at them 10 years later with no apparent change in labeling.

Good luck!

Martin Wessendorf
-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
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