Hi Shiv,
A few options that may or may not have
been tried;
Turning the EM gain off. Depending
on the intensity of the fluor, you actually may be adding noise. If you
turn the EM gain off, you may get a “better” image, and therefore
you may be able to reduce the exposure/laser power.
Make sure you have optimized the filters
in the path.
Also, I believe you may have additional
optics in the emission path that are stealing valuable photons.
As stated, “30” (FYI this is
in a non-linear scale to 100) is not a very informative number to determine
intensity at the sample as it depends size of laser and throughput i.e. optics
in the excitation path (this is with a CSU22). Your measurement of 240um,
however, is perfectly objective (excuse the mild pun). And, again as
stated, is very reasonable.
Talk soon.
Best,
Gary Laevsky, Ph.D.
Imaging Application Specialist
Andor Technology
discover new ways of seeing
Cell (774)
291 - 9992
Office
(860) 290 - 9211 x219
Fax
(860) 290 - 9566
Web:
www.andor.com
From: Confocal
Microscopy List [mailto:[hidden email]] On Behalf Of Mayandi Sivaguru
Sent: Monday, November 24, 2008
6:09 PM
To:
[hidden email]
Subject: Photobleaching of GFP in
Spinning Disk Confocal
Hi all, One of our client is using a cell line expressing GFP bleaches very
fast (Andor Revolution SDC system), despite using low excitation setting at the
488 line laser (set at 30 at the AOTF and approximately 240 microW on the 100x
Olympus UPLANSAPO objective), EM Gain in combination with high amplifier gain.
She wants to get two frames per second under two channels she is getting it but
the cell bleaches within couple of minutes. Using live cell antioxidants such
as Trolox is not an option, I would greatly appreciate any insights in this
regard. Will neutral density filters help?
Thanking you in advance
Shiv
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