Choosing filter configurations for Qdots in Flow

Nick Holmes nh106 at cam.ac.uk
Fri Jun 15 11:59:27 EDT 2007


In message <BEF98223DBEEBC48994F8118FEDB6F2D0131D7C6 at rex1.intranet.epf 
l.ch>
	  "Roberts Joanna" <joanna.roberts at epfl.ch> wrote:

> 
> 
> Dear Flow Cytometry Community,
> 
> 
> 
> I have a question about filter configurations for Qdots in flow
> cytometry.
> 
> 
> 
> I would like to order a new instrument with a violet laser to measure
> the full range of qdot fluorochromes. From quick talks with BD, I have a
> list of the filters they suggest. They represent slightly narrower
> signal ranges in just about all cases than the filters used by the
> fellows in the paper by Chattopadhyay et al in Nature Immunology last
> year.
> 
> 
> 
> For those of you working with these reagents in flow, could you offer
> your opinion on this matter? Would you recommend collecting more signal
> (and probably doing a bit more 'inter-qdot' compensation) or would you
> suggest taking the narrower band pass, collecting less signal and
> needing less compensation?

Dear Joanna

We have not had a lot of experience with Qdots but what we have done 
shows that there are significant differences in average quantum yield 
(QY).  In our samples qdot655 was much brighter than qdot585 (compared 
like-for-like).  If you look at the company's literature I recall that 
they seem to suggest different QY is a general property of specific 
wavelength qdots.  It may be this which is colouring BDs thinking - 
obviously brighter qdots would need lower bandwidth.

However, a paper by Yao et al (PNAS doi 10.1073) suggests that there 
is significant batch-to-batch variation of QY (and that this is 
related to the dark, ie inactive, fraction of particles).

Whatever, they are extremely bright by normal flow standards so 
assuming your samples will have a reasonable labelling density, narrow 
band filters will work perfectly well and make sense by reducing 
compensation issues.


Best wishes

Nick
-- 
Nick Holmes	Division of Immunology, Dept of Pathology
		Cambridge University, Cambridge CB2 1QP, UK
		Tel:+44 1223 333871  Fax:+44 1223 333346
mailto:nh106 at cam.ac.uk	http://www-immuno.path.cam.ac.uk/~nh106/



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