Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

While this would be true for multi-photon photobleaching, this is not the
case for single photon photobleaching. The extent of photobleaching above
and below the focal plane will depend on the NA of the objective lens and
the size of the ROI.

First consider the case of a stationary beam (i.e. non-scanning, as in the
first FRAP experiments). The photobleached volume will directly be
proportional (approx. inversely exponential, not linear) to the point spread
funtion of the exciting beam. In this limiting case, there will be very
little bleaching above or below the focal plane because the excitation
intesity is very low there. Evidently, this depends on the NA of your
objective lens: the higher the NA, the faster the intensity drops along the
optical axis further from the focal plane (because the illumination cone is
wider and hence less photons per unit area).

However, when bleaching a 2D ROI with a scanning beam, the decrease in
intensity along the optical axis is counteracted by the increase in
effective illumination time for those points. While this is difficult to
explain in words, it is immediately clear from a simple intuitive figure as
can be found in the second edition of the Handbook in Chapter 34 or 36 (I
forgot which one and don't have the book here with me; maybe the figure is
in the 3rd edition as well, but I didn't check). Let me know if you would be
interested in calculating this more precisely.

In reality, things are even more complicated, because photobleaching does
not simply depend on the number of photons, but on the photon flux, i.e. the
number of photons per unit area and unit of time. Generally, more
photobleaching will occur when the same number of photons are applied over a
longer period of time. This is because photobleaching requires chemical
reactions to proceed with a finite reaction speed. At a certain point
photons will arrive faster than the reactions can proceed and cannot cause
further photobleaching.

Hope this helps,

Best regards,

Kevin

Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: +32 (0)9 264.80.78
Fax: +32 (0)9 264.81.89
E-mail: [log in to unmask]
 

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List 
> [mailto:[log in to unmask]] Namens Ghafar Sarvestani
> Verzonden: woensdag 28 maart 2007 11:56
> Aan: [log in to unmask]
> Onderwerp: Photo bleaching (FRAP)
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Dear Confocalist,
> Can you help me out on this FRAP question. Is it right in 
> thinking that if you focus bleaching, on a small ROI on a 1 
> micron thick section of the membrane, the parts of the 
> membrane above and below that section are not bleached. In 
> other word when you do the bleaching is that focal plain of 
> the cell or does it bleach throughout the cell from top to 
> bottom in that area?
> Any advice you can give would be most appreciated.
> With regards,
> Ghafar
> ***********************************************
> Ghafar T Sarvestani, PhD
> Hanson Institute for Medical Research
> Hanson Building level 2, Room 2-42-N
> IMVS, Frome Road, Adelaide SA 5000
> 
> Affiliate Lecturer
> Department of Medicine
> The University Of Adelaide
> Tel: +61 8 82223603
> Fax: +61 8 82223162
> Email:[log in to unmask]
>