Hi Zoltan
I think using a short wavelength could be
the problem. Not only will you induce more phototoxicity compared with longer
wavelengths but you will also bleach your FRET acceptor and donor which presumably
absorb blue light. One solution if you have a 633/640/647 laser is to label
your nuclei with a far red fluorophore for live cells. You could use Draq5 http://www.biostatus.com/product/draq5/.
Med vänlig hälsning / Best regards
Sylvie
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Sylvie Le Guyader
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14157
+46 (0)8 608 9240
From:
Sent: 27 March 2009 13:19
To: [hidden email]
Subject: Overnight scan of human
fibroblasts
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
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