Re: Sorting by Chemiluminescence

I've been interested in this for a long time.	We even tried to repeat one of the
published reports (without success).   I think that I can speak for others in my industry
that most of us would line up to purchase a "LACS" instrument if it were available.   The
obvious application is optimization of luciferase reporter cell lines.	 

Phil Marder
Lilly Research Labs.


-----Original Message-----
From: Michael Corey <coreym@targen.com>
Sent: Aug 30, 2004 3:33 PM
To: cyto-inbox
Subject: Re: Sorting by Chemiluminescence

Howard certainly knows much more about flow cytometry than I ever 
will, but doesn't this depend on how you look at it? A couple of 
points: according to my limited understanding a bioluminescent 
"lifetime" is not the same as a fluorescent lifetime--the latter is 
the time to re-emit a single photon, while the former is usually 
defined (I think) as the tau of the luminance signal, which might 
involve turnover of thousands of molecules of ATP or NADH (and 
therefore photons) per luciferase molecule. It's certainly true that 
pumping photons in with a laser is a great way to get lots of photons 
back in a short time, but luminance methods can also be extremely 
sensitive. Is the problem with bioluminescence really fundamental, or 
is it the fact that no one has tried to build a LACS? After all, a 
LACS wouldn't need (1) filters, (2) collimation, or (3) a lamp of any 
kind, and with a dark enough chamber, essentially every photon at any 
angle and any wavelength would be signal. Without the limitations 
associated with the laser optics, the detection path could be much 
longer, which would help to address the "lifetime" issue. It may be 
in-born naivete, but it seems to me that the reason we don't have 
luminescent flow sorting is that people haven't really tried.
Michael

>Tim Kute wrote:
>
>>Has anyone ever tried to sort cells using chemiluminescence as the indicator?
>>
>>An investigator has some cells that have been transfected with 
>>luceriferase and could mix the the cells with the substrate prior 
>>to putting on the machine.  Is there a known protocol for 
>>seperating positive cells from the negative transfected cells?
>>
>>If yes,  Please send me a article or protocol.
>
>Lindqvist C, Karp M, Åkerman K, Oker-Blom C: Flow cytometric 
>analysis of bioluminescence emitted by recombinant 
>baculovirus-infected insect cells. Cytometry 15:207-212, 1994
>
>These authors apparently detected low levels of bioluminescence in a 
>flow cytometer, although the cells to which luciferin was added were 
>measured in a 488 nm laser beam, complicating the issue. In general, 
>bioluminescence lifetimes are very long (seconds to minutes), and 
>one would not expect to collect many bioluminescent photons in the 
>few microseconds it takes for a cell to pass through the observation 
>region of the cytometer. You'd be better off imaging a large number 
>of cells for a much longer time and selecting the bright ones with 
>laser capture or old-fashioned microdissection, because, if you 
>slowed the flow cytometer down enough to get good bioluminescence 
>signals as a basis for sorting, your analysis rate would be one cell 
>every few seconds.
>
>-Howard


-- 
Michael J. Corey, Ph.D.
Associate Director, Assay Development
CellExSys
1100 Olive Way, Suite 100
Seattle, WA 98101
USA
Tel.: 206-521-4846
Fax: 206-521-4841
Email: coreym@targen.com

"Why did the dinosaur cross the road? Because the chicken hadn't 
evolved yet."  --Tomas Corey, age 7




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