Re: Photobleaching of GFP in Spinning Disk Confocal

Posted by Knecht, David on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1576815.html

I would have to respectfully disagree with Gary on this one.  If you are photobleaching GFP, then keep turning down the laser power until it stops.  There is no reason you should see rapid GFP photobleaching with an EM camera.   I have found very little benefit in turning down the EM gain. All that does is decrease the signal without affecting the noise much.  I find that it is far better to turn down the laser (I go as low as 1% on the AOTF for bright GFP cells) and use a 2x or 4x average if the image is noisy.  The EM camera is amazingly sensitive, but noisy.  You gain very little by having high laser power as that camera is designed to detect faint signals and benefits little from a strong signal.  You also have to be careful not to be misled by the autocontrasting the software does to display the 16 bit image.  If you have an intensity that is 100-200 over background, the conversion can make the screen image look poor, but adjusting the brightness/contrast manually can get you a nice looking image.  I go by the relative pixel intensity of cell vs. background rather than the subjective image quality to set up an experiment that is challenging.  Dave

On Nov 25, 2008, at 8:34 AM, Gary Laevsky wrote:

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

 

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From: Confocal Microscopy List [[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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Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

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University of Connecticut

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