Re: Photobleaching of GFP in Spinning Disk Confocal

Posted by Knecht, David on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1579080.html

Interesting observation about Oxyrase.  We found that Oxyrase increased the resistance of cells to phototoxicity when imaging live GFP expressing cells at low intensity where photobleaching was not an issue.  Dictyostelium is very sensitive to light exposure and round up quickly when exposed to fluorescent excitation.  They tolerated the light better in the presence of Oxyrase.  Obviously this whole issue is very complicated!  Dave

On Nov 25, 2008, at 6:04 PM, Beat Ludin wrote:

I actually found that EGFP "bleaches" about 20x faster in the absence of oxygen (e.g. in the presence of Oxyrase)! And it recovers slowly once oxygen is present again. I made this observation (and tried to publish it in vain) before the photoconversion properties of GFP became known so, without having done any further tests, I would now assume that what I observed back then wasn't bleaching in the true sense.
TCLSS, make sure there is oxygen around when you want to image EGFP (I don't know about other variants). If you just put a wet coverslip with cultured cells on a slide, the cells will metabolize the oxygen in no time.

Cheers,  Beat

At 18:49 25-11-2008, you wrote:

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>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>http://www.fhcrc.org/

===

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

Office: 217.333.1214

Fax: [phone]

<[hidden email]>[hidden email]

http://core.igb.uiuc.edu

Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)