RE: 50 mW violet laser

What about using the Coherent Connection software to attenuate the laser to
25mW in the new instrument to match the old instrument.


Of course, that requires a Cube model laser, but I believe that is what BD
is putting into the LSRII by default.


Seems like a simple solution.


-Christopher Bare

Rockefeller University

Flow Cytometry Resource Center


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From: Telford, William (NIH/NCI) [E] [mailto:telfordw@mail.nih.gov] 
Sent: Wednesday, April 16, 2008 2:45 PM
To: cyto-inbox
Subject: RE: 50 mW violet laser


Hi Dave.


Violet lasers diodes in the 50 mW range have been around for a couple of
years now - they should be at least as reliable as the earlier units.
Several manufacturers have ~100 mW units too.


In our experience, having additional power in the violet is a good thing.  A
25 mW unit will work fine for most immunophenotyping applications and
viability (i.e. Cascade Blue, Pacific Blue, Live Dead Violet, etc.).  For
bright labeling, you may not see much practical difference between 25 and 50
mW.  However, having more power should allow better discrimination of weaker
surface labeling from background.  Having more power definitely benefits
fluorescent protein detection like eCFP, Cerulean, etc., especially if you
are using these probes as FRET donors.	We see a noticeable difference in
eCFP s/n ratio as we move up in power.	I think this is why the cytometer
manufacturers have made the more powerful unit a standard item.  I think the
cost difference between 25 and 50 mW units is pretty small now, too.


Enjoy,


Bill




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From: Dunaway, Dave [mailto:Dave.Dunaway@nationwidechildrens.org] 
Sent: Wednesday, April 16, 2008 10:07 AM
To: cyto-inbox
Subject: 50 mW violet laser


Flowmeisters, 

	In an effort to create redundancy across a couple of our LSR II's we
are looking at the addition of a 405 violet laser to one that lacks such.
One of the machines that does have a violet laser has a Coherent 25 mW.  In
a quote the we received from BD they specified a 50 mW laser.  When asked
about the fact that we wanted the 25 mW version the response we received was
that they weren't sure if they still offered a 25 mW version.  That aside,
this situation has raised a couple of questions that I am hoping someone on
the listserve could address.

	Our rationale for wanting the 25 mW rather than the 50 is that
higher power of the 50 will create a greater difference in signals
generated, thereby moving away from our goal of redundancy across machines.
While no two machines will ever be exactly the same, and proper controls and
data analysis would be paramount we want to be prepared to allow our users
to switch between machines in the hopefully rare case of there being no
other choice.  In your opinion does the use of a 50 vs a 25 create more
potential disparity than both machines having 25's?

	Secondly, does anyone out there have experience with the 50 mW
laser?	Is it more problem prone?  I guess the general question would be
what are the pluses and minuses both in terms of performance and reliability
with regard to the stronger laser?  Thanks in advance for your input.

Dave 






David Dunaway 
Flow Cytometry Core Laboratory 
The Research Institute at Nationwide Children's Hospital 
700 Children's Drive 
Columbus, Ohio 43205 
(phone) 614-355-2820 
Dave.Dunaway@nationwidechildrens.org 


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