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Before reading Julio's response, I was going to suggest the same thing. It sounds like the dye concentration is too high and is de-quenching. As you image, some of the DiD begins to photobleach, and the self-quenching effect isn't as strong. Hence the sample appears to get brighter with time. 

I have observed the same thing when using similar lipophilic probes like DiI and DiO at too high a concentration.

Try diluting your probe.

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2011-11-01, at 5:57 PM, Julio Vazquez wrote:

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> 
> We observed something similar with a mitochondria label (mitotracker), that was getting brighter over time. It turned out the person doing the experiment was using way too much dye. After they diluted the dye one hundred fold or more, the dye started behaving as expected. My interpretation is that the dye was self-quenching, and as dye was being photobleached, it was losing its ability to quench itself faster than it was being bleached, if that makes any sense. In any event, diluting it solved the problem. Don't know if this is what you are observing, but its easy to try...
> 
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 
> 
> http://www.fhcrc.org/
> 
> 
> 
> 
> On Nov 1, 2011, at 2:43 PM, Vladimir Ghukasyan wrote:
> 
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>> Dear Colleagues,
>> 
>> We're trying to image myelin and face a strange problem.
>> a. Over the time course the DiD lipophilic dye is getting brighter.
>> When observing the sample after LSM with mercury light an overly
>> bright square can be visualized.
>> b. Signal in green channel from myelin particles is increasing with
>> time as well. Since DiD is supposed to fluoresce in 650 - 750, this is
>> strange. Non-labeled myelin didn't seem to have the same signal. This
>> can't be a cross-talk - we are using spectral separation, sequential
>> scanning (FV1000). Same effect observed on Zeiss 710.
>> 
>> Any suggestions?
>> 
>> Thank you in advance,
>> Vladimir
>> 
>> ==================
>> Vladimir Ghukasyan, Ph.D.
>> Confocal and Multiphoton Imaging Facility
>> Neuroscience Center
>> University of North Carolina
>> 
>> 115 Mason Farm Rd., Bld. 245, Rm. 7109F
>> Chapel Hill 27599-7250
>> NC
>> 
>> Tel.: (919) 966 5807
>> Fax: (919) 966 9605