Anje.

I can't answer the details in your question since I don't do FRAP,
although I do have the same microscope. However, I imagine that those
that do know the details would probably be aided by knowing what
fluorophore you are using. If it is the wrong one the 488 line will
not give you the bleaching you need.


>Hi,
>
>I'm trying to perform FRAP experiments on the CLSM MRC1024 equipped with an
>Ar/Kr and Ar UV laser. Therefore, I use the time course experiment option
>for defining regions of interest and measurement of fluorescence intensity
>in this regions. The problem is that it seems to be only possible to bleach
>the FLASH area with the UV laser. However, I want to observe and bleach the
>area only with the 488 nm line. If I block the UV part of the laser the
>entire experiment doesn't work and no bleaching in the Flash area occurs. If
>I let the UV lines enabled than an strong bleaching of the general
>fluorescence in the sample occurs. In addition, this FRAP experiment are
>only possible if I tick the "leave the shutter open" option in the last page
>of the time course experiment notebook. So, cells are exposed to lots of
>laser light during this experiment....
>
>Does anybody perform FRAP experiments with the Biorad CLSM ? Is there the
>possibility to bleach and observe with the 488 line in this microscope ? Is
>something wrong with the configuration of our microscope ?
>
>In advance, thanks a lot for you help
>
>Anje Sporbert
>
>
>
>Max Delbrueck Centre for Molecular Medicin
>Berlin-Buch
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Mario M. Moronne, Ph.D.
Material and Life Science Div.  M/S 6-2100
University of California
Berkeley Lab
1 Cyclotron Rd.
Berkeley, CA
94720

ph (510) 486-4236
FAX (510) 528-8076
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