excitation and emission

Marty Bigos mbigos at gladstone.ucsf.edu
Sun Nov 13 21:32:02 EST 2005


Hi -

As most responders have pointed out, the excitation efficiency of PE 
is better at 532 nm compared to 488 nm. But the curves only tell part 
of the story.

As many have observed PE used in a flow cytometer gives better signal 
to background with low power 488 excitation compared to higher power. 
Using two lasers tuned to 488 nm at 200 mW, I verified that using 
high power once to exicte makes PE unusable for at least 10 ms (and 
possibly longer). This does not happen with 532 nm excitation (and 
generally with PE tandem conjugates). Supposedly (I'm out of my 
league here) the 532 line excites a different part of the energy 
transfer structure in PE than the 488 line.

Thus you should expect to do better than the curves would indicate.

I have not done a comparison for the tandem dyes using the same laser 
powers for 488 and 532 nm; anecdotally 150 mW of 532 nm gives 
excellent results with PE-TR, PE-Cy55, PE-Cy7.

Note that the above does not apply to PerCp or PerCp-Cy5.5.

Marty
Marty Bigos
Director, Flow Cytometry Core Laboratory
Gladstone Institute of Virology and Immunology

Mail:
1650 Owens Street
San Francisco CA 94158
(tel) 415-734-4821
(fax) 415-355-0855
(mobile) 415-845-8450

mbigos at gladstone.ucsf.edu
http://www.gladstone.ucsf.edu
http://www.gladstone.ucsf.edu/gladstone/php/?sitename=flowcytometry

>Look at the excitation curve again, mark. Your green laser will do 
>the job better than a blue one!
>
>
>Frederic I. Preffer
>MGH -Center for Regenerative Medicine
>CPZN -Room 4226
>185 Cambridge Street
>Boston, MA 02114
>
>617 726 7481  v
>617 724 3164  fax
>
>From: Mark D'Amico [mailto:MDAmico at panaceapharma.com]
>Sent: Friday, November 04, 2005 3:16 PM
>To: Cytometry Mailing List
>Subject: excitation and emission
>
>Howdy,
>
>I have a simple, if not outright stupid question.
>
>I have a 532nm laser for excitation. I am using R-PE as my marker. 
>The excitation peak is at 488nm. Is my laser wavelength too long?
>
>Mark D'Amico, Ph.D.
>Scientist
>Panacea Pharmaceuticals
>207 Perry Parkway    #2
>Gaithersburg, MD   20877
>(240) 243-8000 Ext. 206
>mdamico at panaceapharma.com
>


-- 
-------------- next part --------------
HTML attachment scrubbed and removed


More information about the Cytometry mailing list