Re: Photobleaching of GFP in Spinning Disk Confocal**Correction**

Posted by Gary Laevsky-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1581365.html

Shiv,

 

I have been informed I neglected to mention a very important issue.

 

Not only should you turn the EM gain off, but you should also switch to the conventional register.

 

The EM register, because of it’s amplification characteristics adds a tremendous amount of noise to the image.

 

Some EMCCDs allow you to be able to switch to the conventional register.  Your noise will be greatly reduced, and you can then possibly turn down the laser.

 

 

Best,

 

Gary Laevsky, Ph.D.

Imaging Application Specialist

 

Andor Technology

discover new ways of seeing

 

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Cell         (774) 291 - 9992

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Web:       www.andor.com

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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

Mayandi Sivaguru, PhD, PhD
Microscopy Facility Manager
8, Institute for Genomic Biology
University of Illinois at Urbana-Champaign
1206 West Gregory Dr.
Urbana, IL 61801 USA

Office: 217.333.1214
Fax: [phone]
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