Re: Photobleaching of GFP in Spinning Disk Confocal

Posted by Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1577424.html

Hi Shiv, 

Anything that reduces illumination intensity at the sample will help. If you don't need ultimate resolution, you could try binning or using a lower power objective. If your samples are in water, you may try using a water lens if available to reduce spherical aberration, and hence increase detection efficiency. Also, there may be other trivial reasons for the bleaching, such as suboptimal pH in the sample/medium, and, of course, GFP is very sensitive to solvents (nail polish, etc...).  You mention Trolox is not an option. How about Oxyrase:

http://www.oxyrase.com/oxyfluor.html

There was a thread on this list a while ago about the merits of Catalase/Glucose Oxydase

10mM -mercaptoethanol, 2.5 mg/ml glucose, 20 g/ml catalase, 0.1

mg/ml glucose oxydase

The concentrations above are from Chabrillat et al, Molec Biol of the

Cell 16, 1640-1650 (2005).

I think there is still some debate as to whether this actually works...

Julio.

--

Julio Vazquez, 

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

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On Nov 24, 2008, at 3:08 PM, Mayandi Sivaguru wrote:

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

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