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Hi David,

For FRAP your background region should be best within the same field of
view. Moreover it should be in a region that has the same diffusion constant
as your bleached area.

Also note that you cannot infinitely correct for bleaching during recovery.
Bleaching during recovery should not be more than 10-20%, although it
depends a bit on the specific FRAP model you're using. If you have too much
bleaching during recovery, even after correction your recovery curve will be
too much deformed to obtain reliable diffusion constants.

If you have too much bleaching, try lowering the monitoring laser power,
lowering the zoom setting or decreasing the sampling rate. 150 images seems
an awful lot, but of course it depends on the S/N ratio how much images you
need and at what sampling rate to get a reliable recovery curve.

Best regards,

Kevin

-----Oorspronkelijk bericht-----
Van: Confocal Microscopy List [mailto:[log in to unmask]] Namens
david.depoil
Verzonden: vrijdag 24 juni 2005 10:22
Aan: [log in to unmask]
Onderwerp: Re: Correcting for background bleaching

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Hi mike,

I am doing FRAP experiment so I must acquire 150 images every 1,5seconds. I
use a 63X objective. I also work with a zoom 2 in LSM software. I have an
important background bleaching.

Thanks a lot for your answer

David Depoil
>PhD student
>Valitutti's Team
>U563 CPTP Inserm
>Toulouse
>France


A 18:04 23/06/2005 -0700, vous avez écrit :
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi David,
>
>At what magnification are you working?  How long between acquisitions?
>Do you have an automated X-Y stage at your disposal?  
>
>In the past, in long-interval timelapse experiments, I have used an 
>automated stage and I captured an image in an empty field for every 
>image I acquired of my sample.  I then did a frame by frame subtraction 
>of the image sets.  This seemed to work well to get rid of the artifact 
>caused by the "bleaching" of the media over time.  While not as elegant 
>as a mathematical solution, it did the job.
>
>Cheers!
>Mike
>