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I cannot immediately produce any papers on either side of this debate, but I must say it seems reasonable within a certain range of excitations.  It seems quite improbable that it could apply if one is driving fluorescence to saturation.  With 3 or 4 orders of magnitude difference between point irradiance in confocal vs widefield, I would think that it is pushing linearity to its limit.  

Wavelength is also an issue - the more the excitation wavelength is shorter than the optimum for S1, the greater the probability of transitions to higher states, and this is well documented to increase the rate of bleaching.

                                                                         Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Shigeo Watanabe
Sent: Friday, 27 September 2013 12:29 PM
To: [log in to unmask]
Subject: Photobleaching of confocal microscopy

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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