Strange observation

[simon walker (BI)]

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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 

[Feinstein, Timothy]

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Hi Simon,

In my experience 405 nm bleaches everything to some degree and in general
red FPs bleach more easily than green.  I cannot speak for the latest gen
of red proteins but it seemed to be the case when I compared dTomato with
GFP, cerulean, venus etc.  Without checking photostability measurements I
would guess un-optimized dsRed bleaches fairly fast.  If red monomers
bleached slightly faster than green it would remove the FRET effect that
usually quenches green subunits.  Are you absolutely committed to dsRed?

In general I d strongly recommend 442 nm excitation for CFP, but I
understand that is rarely done as you usually cannot run 442 and 405 nm
together and 405 is more generally useful.

Cheers,


Tim

Timothy Feinstein, Ph.D. | Manager, Core for
Confocal Microscopy and Quantitative Imaging
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: [phone] | Email: [hidden email]







On 12/19/14, 7:27 AM, "Simon Walker" <[hidden email]> wrote:

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>posting.
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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://scanmail.trustwave.com/?c=129&d=4pmU1NDdf4kf-ALuJZ1wwYYQXFAMDBNlSvf
>eBH0Uvw&u=http%3a%2f%2fwww%2ebabraham%2eac%2euk%2fscience-services%2fimagi
>ng