[Cytometry] 532 vs 568 laser

Telford, William (NIH/NCI) [E] telfordw at mail.nih.gov
Fri Feb 6 01:36:53 EST 2009


Hi Chris...

I agree with Marty's comments - just to provide our own experience:

(1)  When we do a 532 nm versus 561 nm comparisons using power-matched
lasers (50 mW) and the same optical path/PMTs/detection filters,
excitation of PE is very similar for both.  You do need to use a
slightly longer PE filter with the 561 nm (590/20 nm), which causes a
small loss of PE sensitivity.  We have not looked specifically at the PE
tandems, but I would expect that the 532 nm laser at 150 mW would work
better than the 561 nm 50 mW as Marty sees in his comparisons.  The 561
nm might also be exciting the Cy5 and Cy5.5 portion of the PE tandem to
some extent, which could also be contributing to the signal spreading
Marty sees.  532 nm lasers are available at higher power levels than 561
- the current commercial max for 561 nm is 100 mW.

(2)  We also see the 561 nm working better for mCherry and the longer
reds - however, both lasers work well for DsRed and the other shorter
reds.

(3) The 561 nm laser indeed causes unwanted excitation of APC - this
causes a crossbeam compensation issue with PE-Cy5.  Our comparisons show
that the compensation required between APC and PE-Cy5 using the 561 nm
is roughly 2-5 times more what you will see with the 532 nm.  This is
manageable, but has to be kept in mind when planning multicolor
analysis. 

As far as deciding which laser to go with, for me it's a tough call.  If
you plan on mainly using your system for PE/PE tandem enhanced
excitation, the 532 nm is probably better - it is a well-characterized
system.  If you plan on using your system to do longer red FPs as well
as PE, the 561 nm may be a better option.  We use both lasers in our
lab, and ideally I would like a system with both lasers on it - they are
both very useful.  

In our hands, both 532 and 561 nm lasers show excellent reliability as
well.

Enjoy,

Bill Telford
NCI-NIH

-----Original Message-----
From: Christopher Norbury [mailto:ccn1 at psu.edu] 
Sent: Sunday, February 01, 2009 10:10 AM
To: Flow list
Subject: [Cytometry] 532 vs 568 laser

Folks,

We are hoping to begin purchase of a new 4 laser instrument in the near
future.  We've settled on 3 lasers to include, a 405nm near-UV, the
standard
488nm and a 633nm.  We are now deciding between a 532nm and 568nm laser
for
the mid-ranges.  Could anyone give any input on the applicability of
these
lasers for various dyes (PE, PE tandem conjugates, fruity dyes (mCherry,
tomato etc), RFP etc?  Also, is there any major difference in the
reliability of these two respective lasers?  Finally, any information on
the
compensation issues of the 532 with the 488 and the 568 with the 633
would
be much appreciated.

Thanks


Chris


-- 
Chris Norbury, Ph.D
Associate Professor of Microbiology and Immunology
Scientific Director, Flow Cytometry Core Facility.
Penn State Milton S. Hershey College of Medicine
Room C6804A, Dept. of Microbiology and Immunology, H107
500 University Drive, Hershey, PA 17033-2360
 
Lab      717 531 0624
Office   717 531 7204
Fax      717 531 6522
Email    ccn1 at psu.edu
Web      http://fred.psu.edu/ds/retrieve/fred/investigator/ccn1

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