RE: Near UV laser in FACS Aria II

Hi Bill,

	Right - the 375 nm laser is essentially as you describe; but as 
far as Indo-1 not working goes, don't forget that ratiometric Ca++ 
detection with a single loaded dye may be nicely done also with Fura Red 
using two excitation lines (same emission near 670 nm). In fact, as I 
recall the Fura dye was the preferred Ca++ dye before the Indo-1 method, 
and it took some time for Indo-1 to be accepted. The old alternative 
'combo' method using fluo-3 with Fura Red was devised simply because there 
were no violet lasers in those days on benchtops - you don't need the 
fluo-3 if you have the standard violet laser (ie, violet excited Fura Red 
replaces 488 excited Fluo-3 for the hi Ca++).

	Joe 



Joseph Trotter
Director, Cytometry / Advanced Technology Group

BD Biosciences
tel: (858) 812-8946  cell: (858) 449-7338  fax: (858) 812-8979
10975 Torreyana Road, San Diego, CA 92121-1106 USA 
E-mail: Joe_Trotter@bd.com   Website: www.bd.com




"Telford, William (NIH/NCI) [E]" <telfordw@mail.nih.gov> 
02/25/2008 11:10 AM

To
Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
cc

Subject
RE: Near UV laser in FACS Aria II






Dear Maayan...

Mark Kukuruga or someone from BD can correct me if I mess up the details
- but as I understand it, the 375 nm laser does not replace the violet
on the Aria II, but is installed as an additional laser.  It is mounted
separately from the fiber-coupled lasers, and does not use a fiber optic
but is reflected into the flow cell from a position downstream from the
fiber outputs.		 But it does use the same flow cell pinhole and 
PMT
trigon as the violet - so you would probably turn off the violet laser
when using the 375 nm.

The main purpose of the 375 nm is to do Hoechst side population
detection of stem cells and progenitors, which does not always work well
with the violet laser.		 The 375 nm will also excite quantum dots, 
but
the violet does this well too.		 The 375 nm will also do a 
somewhat
better job of DAPI cell cycle analysis, although again the violet works
tolerably well for this application too.  Unfortunately, the 375 nm does
NOT do indo-1 calcium detection, since the calcium-bound emission from
indo-1 (~390 nm) is too close to the laser emission.  And the 375 nm
cannot replace the violet laser for good Pacific Blue, Live/Dead Aqua,
etc. excitation - it just doesn't excite these violet-excited probes
well enough.  So the main reason for getting a near-UV diode would be to
do Hoechst SP. 

Enjoy,

Bill Telford
NCI-NIH


-----Original Message-----
From: Maayan Duvshani-Eshet [mailto:duvshani@techunix.technion.ac.il] 
Sent: Sunday, February 24, 2008 1:40 PM
To: cyto-inbox
Subject: Near UV laser in FACS Aria II


Hello everyone,

I ordered the BD FACS Aria about 4 months ago, knowing that I will
receive
the Aria-II. I ordered it with 3 lasers: blue, red and violet, and now I
saw
that the Aria-II can come with a near UV laser (375 nm). Assuming that I
can
change the Violet laser with the near-UV laser; can anyone advise me
what
will be the advantages of the Near UV laser over the violet (405 nm) in
terms of biological applications?

Thank you all in advance


Maayan Duvshani-Eshet, PhD
Infrastructure unit 
The Lorry I. Lokey Interdisciplinary Center for 
Life-Science and Engineering 
Technion - Israel Institute of Technology
Haifa, Israel, 32000
Tel: 972-4-8295834 
Fax: 972-4-8225153
Email: duvshani@tx.technion.ac.il







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