I think as already stated before in this discussion that as long as your bleaching phase is short in comparison to the half time of recovery you observe (e.g. < 5%) that the measurements can be quite good.
 
If I read you mail correctly you are doing Strip bleaching experiments and then the difussion constant should be one dimensional when using single photon excitation.
 
So the question is now what is your half time of diffusion in comparison to the time interval. I am doing FRAP experiments myself on Histone 2B and then multiple bleach itterations are usefull since they are always very short in comparison to the half time of diffusion (0,5 s against 400s) But for bleaching studies with for example free GFP you should use only one itteration in principle.
 
In response to James Pawley, it is true damage can be caused but only when using very high laser power with numerous itterations so as long as you can prove your cells are still viable after the experiment do not worry about that too much.
 
Joost willemse 
Dept. Molecular Biology
Wageningen University and Research Centre
Dreienlaan 3  
6703 HA Wageningen

	-----Original Message----- 
	From: Confocal Microscopy List on behalf of Narasimham Jammi 
	Sent: Thu 25-11-2004 23:00 
	To: [log in to unmask] 
	Cc: 
	Subject: Re: FRAP experiments
	
	

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	>
	> Next to this I have questions on Jammi and Kevin:
	> 1) what confocal do you use?
	> 2) do you know your switching time between bleaching- and recovery phase?
	> 3) what FRAP model do you use?
	> 4) what parameters do you take into acount in your model?
	> And probably there will be many many more questions....
	>
	
	
	Hi Guido,
	1) I am right now performing my FRAP experiments on  a LEICA SP2 AOBS
	confocal
	2) I am doing my bleaching and recovery in the FLY mode...so it's a
	bidirectional scan..my time interval between scans is about 200 millisecs.
	3 and 4) As for the FRAP model, I have been using a 1 dimensional lateral
	diffusion model (although I am not sure this is what i have/need to use)
	
	-Jammi
	p.s thanks Kevin and the others for all your input..
	happy turkey day all!