Re: Photobleaching of GFP in Spinning Disk Confocal

Posted by Beat Ludin on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wasabi-freeware-tp1574097p1578586.html

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]
>><mailto:[hidden email]>[hidden email]
>>http://core.igb.uiuc.edu