[Cytometry] 532 vs 568 laser

Marty Bigos bigos at stanford.edu
Sun Feb 1 19:30:16 EST 2009


Hi Chris -

I had to make the same determination for this past month for new instrument purchase.

I compared 150 mW 532 nm laser on an LSR-II with a 50 mW 561 nM laser on an Aria II. Although the detection filters were the same except for PE on both instruments, the excitation geometry and PMTs are different. So this comparison is not optimal, but did give some useful (I think) results.

The following were evaluated on both instruments: HELA cells with and w/o mCherry, antibody capture beads stained with PE, PE-TR, PE-Cy5, PE-Cy7, APC, and Q-Dot 605, and our standard Spherotech beads.

Both instruments were set up using CST and checked with the Sphero beads. These beads had signal levels at the same orders of magnitude on both machines.

The data was evaluated using SI (Staining Index) as the comparison.

Even with the laser power differences, the SI on mCherry and the PE-tandems was superior with the 561 laser. It was not as good with PE, which is to be expected since the PE bandpass with the 561 laser is narrower.

However, when compensated, PE and all its tandems excited with 561 nm resulted in significantly greater spreading on the compensated measurement parameters, thus resulting in larger SI than with the 532 laser resulting in less sensitivity. Visually this showed up as a higher signal level for the upper edge of the compensated signal, sometimes being a half decade higher.

Moreover APC was more efficiently excited by the 561 laser, and had higher backgrounds when the compensated signals were looked at in PE-Cy5.

The one anomalous measurement was Q-dot 605. I expected it to be excited better by the 532 laser; in fact it was better excited by the 561. Correspondingly, the compensated Q605 in PE-TR had less spread with the 561 laser than the 532 laser. I don't have any explanation for this.

BD is now offering a 100 mW 561 nm laser; the extra power may, except for the APC PE-Cy5 result, reverse some of the above I obtained. However the quotes we received indicated the 100 mW cost more, while prices for systems with the 50 mw or the 532 laser were equivalent. I have had no problems with reliability or stability of the 532 nm laser.


Marty

----- Original Message -----
From: "Christopher Norbury" <ccn1 at psu.edu>
To: "Flow list" <cytometry at flowcyt.cyto.purdue.edu>
Sent: Sunday, February 1, 2009 7:09:33 AM GMT -08:00 US/Canada Pacific
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
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