Zoltan,

As was pointed out, the shorter the wavelength, the more the damage,  
and therefore 405 nm is probably not the best choice for long term  
imaging., although certainly better than UV. Shifting to a red  
nuclear dye would make a big difference.

This being said, you also need to exclude trivial reasons. I don't  
know how much power 20% is on your system (depends on the lasers you  
are using and the overall efficiency of the system), but 20% power  
for a 514 line seems very high to me, if you are using "standard"  
lasers with about 10 mW output. With our Argon laser, we typically  
use 2%-3% power for live imaging, and that is generally plenty. Even  
with our wimpy 1 mW 543 nm HeNe (basically a laser pointer), we often  
need no more than 10%. Therefore, I would recommend you check the   
efficiency of your system and maybe the alignment of your optics.  
It's amazing how a slight misalignment of the pinhole can lead people  
to crank up lasers to lethal levels...


--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

http://www.fhcrc.org

==






On Mar 27, 2009, at 5:18 AM, Zoltan Cseresnyes wrote:

> Dear All,
>
> One of our users would like to scan cultured human fibroblasts  
> (focusing only on the nucleus) for about 15-17 hours at a time, 6  
> um Z stack (7 steps), 3-5 min time resolution, in 5% CO2 at 37C.   
> We have done a few very successful experiments this way already,  
> using our SP5 inverted with an environmental chamber (The Box).  We  
> used 458 nm or 488 nm excitation in those successful experiments at  
> low laser power (<=20%).  Last night we tried 405 nm excitation  
> (20% laser power) as well because we were attempting to do a live  
> FRET scan; the scanned cells all died after about 2 hours.  Do you  
> have any suggestions about how to protect these cells from  
> photodamage for 15-17 hours?  We are trying to keep our lasers low,  
> we were doing 20% power on both the 405 and 514 nm lines.  The  
> temperature is fairly stable at 37C (+-0.8C perhaps) and the CO2 is  
> okay as judged by the colour of the phenol red and the excellent  
> survival of the cells in the non-scanned regions.    In one  
> experiment last week we used 458 nm at 48% power (we had a poor  
> fluorescence signal that time but we tried anyway) and then the  
> scanned cells also died after about 6 hours.  These findings seem  
> to point towards photodamage, hence my enquiry to this excellent  
> forum of experts!
>   Thanks very much,
>
> Zoltan
>
> -- 
>
> Zoltan Cseresnyes
> Facility manager, Imaging Suite
> University of Cambridge, UK