Re: ECFP analysis on Vantage with 457nm line (secondary laserline)

Hi Alex,

We run ECFP off the 457nm line of our I-70 all the time but we do not 
use the 488nm at the same time.  The maximum emission of ECFP is very 
close to 488nm so as I see it your choices are

1)Use the 514nm line of your primary laser to trigger your FSC (if 
you are not using an Enterprise laser).  This way the 514nm line will 
not interfere with your collection of ECFP emission.  Make sure to 
take out the 488nm filter in front of the FSC detector and replace it 
with something that will allow 514nm through but restrict as much as 
possible all wavelengths below 500nm where the emission of ECFP is 
strongest.
2)If you must use 488nm as your primary line put a 488nm 
restriction/blocking bandpass filter in front of your ECFP filter. 
You should buy a filter with as narrow a band around 488nm as 
possible so as not to lose to much of your ECFP signal some of which 
will be blocked by your 488nm restriction filter.  Try to purchase a 
filter with an optical density (OD) of 5 from 483-493nm.  I have used 
the 485/22 filter BD supplies with the instrument many times for ECFP 
collection but I would fear to great a loss of signal with a 488nm 
restriction filter in front of a 485/22.

For system alignment I really like the 8 peak Spherotech beads.  They 
give very nice signal when excited at 457nm and help you control for 
background.  You should be able to easily see a difference in signal 
from the 1st and 2nd lowest intensity peaks.  If you can not you 
either have a bad system alignment or you have high background from a 
laser line leaking light into your collection detector (i.e. 488nm 
leaking light into your ECFP detector).

Good luck and let me know if I can help.

Glenn


*************************************************
Glenn Paradis
MIT Flow Cytometry Core Facility
E18-463
Massachusetts Institute of Technology
Cambridge, MA 02139
(617)253-6454 lab
(617)452-4132 office
(617)253-3714 fax
gap@mit.edu
http://web.mit.edu/flowcytometry/www/
*************************************************




>Dear Flowers,
>
>On a FACSVantage SE:
>Is anybody using the 457 line of a Spectrum 70 laser  as a secondary 
>laser to excite ECFP ?
>The primary laser is 488nm.
>What is the best filter ?  470/15 , 485/22
>Do I need other filters (eg. blocking filters ?)
>Which beads can be used for the laser alignment ?
>It would be great if you could share your experiences with me.
>Many thanks
>Alex
>
>
>--
>______________________________________
>Dr. Alexander Schmitz
>FACS Core Facility
>Department for Molecular Biology (MBI) C.F. Moellers Allee 130/3
>DK 8000 Århus
>Denmark
>axs@mb.au.dk
>Tel: +45 8942 2676
>Tel: +45 2338 2429 Mobil
>Fax  +45 8916 6500
>______________________________________

-- 
Glenn Paradis