Posted by
simon walker (BI) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-observation-tp7583174.html
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Dear List,
I made a strange observation yesterday which I'm hoping someone might
have an explanation for. I was imaging live cells expressing CFP, GFP and
DsRed. The confocal was configured in a sequential line scanning mode to
minimize bleed through, using 405, 488 and 561 nm excitation for the
different FPs respectively. I noticed a strong green signal coming through
from the DsRed (labeling mitochondria in this instance) in the GFP
channel. Ok, so you might think this isn't so strange as DsRed is known to
mature through a green intermediate, but the weird thing was, if I turned
off the DsRed and CFP channels, and imaged the GFP channel alone, then
the bleedthrough disappeared. Moreover, I found that if I was capturing all
the channels together (still in a sequential line scanning mode), turning off
the 405nm laser also eliminated the bleedthrough. The bleedthrough was
only seen in cells expressing DsRed, so my interpretation is that somehow
the 405nm excitation is causing a temporary change in DsRed such that it
can be excited at 488nm, with emission in the green part of the spectrum.
Has anyone else seen this?
Thanks,
Simon
Imaging Facility
Babraham Institute
Cambridge, UK
http://www.babraham.ac.uk/science-services/imaging