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Ghafar,
   If the fluorophores are present throughout the depth of your sample, you will bleach some right through.  The exact shape of the bleach volume will depend on the objective lens, but is very approximately a double cone with the "sharp" ends at the focal plane; the highest bleach efficiency is here, and the efficiency decreases at planes further from this.

But I am not sure what you mean by "1 micron thick section of the membrane"; cell membranes are <10nm thick.  If you bleach while focussed at the top surface of a cell, and there are no fluorophores in the cytoplasm, then only this ROI will be bleached - the lower surface of the cell will be almost unaffected when using a 60x or 100x lens.  This is the original FRAP method.  If you bleach a very small or thin cell such as the tail of a sperm cell, or the edge of a cell where the membrane is "vertical", then you will affect fluorophores at positions out of the focal plane.

Ian  

------------------------------Dr. I.E.G.Morrison       [log in to unmask]
                              Dept.Biological Sciences, University of Essex
                              Wivenhoe Park, Colchester, Essex CO4 3SQ
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]On
Behalf Of Ghafar Sarvestani
Sent: Wednesday, March 28, 2007 10:56 AM
To: [log in to unmask]
Subject: Photo bleaching (FRAP)


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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
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Ghafar T Sarvestani, PhD
Hanson Institute for Medical Research
Hanson Building level 2, Room 2-42-N
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