*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Shigeo,

it definitely is not so simple. Beyond certain intensity threshold the 
fluorescence intensity is not proportional to illumination intensity due to 
e.g. fluorescence saturation or dark (triplet) state formation. In this case
also the absorption is lower and the relative rate of photobleaching may 
also decrease. This however depends on such effects as dark state absorption
etc.

It is surprisingly common to achieve these conditions in confocal imaging as
the local illumination intensity in the focal spot is very high.

You may find more info e.g. here:
ROLF T. BORLINGHAUS, MICROSCOPY RESEARCH AND TECHNIQUE 69:689–692 (2006)
Gerald Donnert, NATURE METHODS 4 (2007) 81

Regards,

zdenek svindrych



---------- Původní zpráva ----------
Od: Shigeo Watanabe <[log in to unmask]>
Datum: 27. 9. 2013
Předmět: Photobleaching of confocal microscopy

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Listers,

I have a basic question about the relationship between photobleaching, 
pixel dwell time and intensity of excitation beam in confocal microscopy. 

Review paper "Optical Sectioning microscopy" in Nature Methods 2005 said 
about bleaching of confocal microscopy.

"The probability that a molecule bleaches depends on its exposure to the 
excitation light. This is the 
product of the irradiance a molecule receives and the time it receives it. 
Thus, a molecule that receives irradiance I1
of duration t1 is as likely to bleach as one that receives twice the 
irradiance (2I1) for half the time (t
1/2)."

Although this is well accepted among confocal microscopy user, I am 
wondering if there are any data or paper to support this notion.
I also would like to know how exactly same the photobleaching of these two 
cases are.

If someone knows any information, we appreciate comments.

Best Regards,
Shigeo Watanabe, HPK"