Posted by
Jordan Becker on
URL: http://confocal-microscopy-list.275.s1.nabble.com/mEos4-photoconversion-tp7583802.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Hey I hope somebody can help me out. I've started working with a fusion
protein of mEos4a and the photoconversion has been working poorly. I'm using
a swept field confocal and Prairie View software.
I am using a 488 laser at 20% power and 561 at 35% power for visualizing the
protein in live cells. I notice that the green form of mEos bleaches quite
quickly even as I'm identifying cells of interest to image. In addition,
there tends to be quite a bit of red signal already detectable in these
cells without photoconversion by a 405 laser.
I am able to get ~500% increase in red signal and maybe a 50% decrease in
green signal upon 405 conversion (but still far from complete and it was
difficult to find cells with minimal red signal). I converted using 20 fast
repetitions of a relatively low powered 405 (~10%) at a single point
(0.500micron diameter).
Does anyone have any advice for best visualization laser and photoconversion
laser settings? I'm looking to use mEos4 for FRAP/FLAP. Is mEos sensitive to
ambient light
Best,
Jordan