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Hi all,
When discussing strange events that occur during
FREAP, I think that we need to remember that when
Richard MacIntosh did careful TEM studies on what
the insides of cells looked like after they had
been exposed to enough light to "beach" the
fluorophor, the local internal structure of the
cells had been destroyed in the bleached region.
i.e., Perhaps the GFP got brighter because it was
released from the protein it was supposed to be
tagged to. Or was just more able to move freely
in the milieu, and so more often had its dipole
axis line up with the pol direction of the
excitation. Or destruction of organelles having
high RI (like mitochondria?) made the spot closer
in shape to the one described by Airy? Or the
increase could be part of a wound-healing process
initiated by a cell sadly unaware that we are
expecting it to "take-it-like-a-man" and continue
as if it had NOT just had its insides scrambled.
Cheers,
Jim Pawley
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>
>Hi, we have observed a bit different effect when
>doing FRAP on GFP. We used 488 nm for imaging
>and 405 nm for bleaching. After a brief
>bleaching the GFP signal increased considerably.
>We observed this in samples that also showed
>fast initial photobleaching of GFP during
>pre-bleach recording. So we concluded the GFP
>goes to some dark state during pre-bleach
>measurement and is re-activated with brief 405
>nm illumination. The 488 light was probably too
>strong... Best, zdenek ---------- PÛvodní zpráva
>---------- Od: Julio Vazquez
><[log in to unmask]> Komu:
>[log in to unmask] Datum: 19. 9.
>2014 23:29:07 PÞedmût: Re: Increase in cell
>brightness after FRAP "***** To join, leave or
>search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include
>the link in your posting. ***** Not sure this is
>the answer to your issue, but we had something a
>bit similar once with one of our users. In their
>case, they were using a mito- tracker dye to
>look at mitochondria. Signal kept going up the
>more we imaged. Don't remember if we were doing
>FRAP or just imaging. After much head
>scratching, I told them to do a dilution of
>their label. It turns out they were using about
>1000x times more than what they needed and my
>explanation is that the dye was self-quenching;
>by "bleaching" it, we were somehow reducing the
>quenching and getting an increase in signal.
>When they used a 1000 fold dilution ( or at
>least a few hundred fold), the signal was still
>high or higher than when they were overloading,
>and no increase was seen during imaging. Doing a
>dilution series with GFP is not quite as
>trivial, but I wonder if they are seeing similar
>issues due to gross overexpression of their GFP?
>Julio Vazquez Fred Hutchinson Cancer Research
>Center Seattle, WA 98109
>http://www.fredhutch.org On Sep 19, 2014, at
>1:43 PM, Wendy Salmon wrote: > ***** > To join,
>leave or search the confocal microscopy
>listserv, go to: >
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >
>Post images on http://www.imgur.com and include
>the link in your posting. > ***** > > Hello
>all, > > I am hoping that the vast experience
>and expertise here might have an answer to a
>strange phenomenon one of my users is seeing
>with FRAP on my Zeiss 710. She is FRAP-ing ~2um
>diameter region in cells expressing GFP- fusion
>proteins with close to diffusion rates. > >
>After the regional bleach event and diffusion
>recovery, many of the cells exhibit a
>significant increase in total intensity over the
>starting intensity. Some of the time-intensity
>plots make it look like the recovery is 150% or
>more! Imaging cells without the bleach event
>never results in this signal increase. > > Has
>anyone seen this before and have suggestions on
>what might be going on? > > The FRAP settings
>are 40x/1.3NA oil objective with 100% power of
>the 488 Argon at scan speed of 2 for 1 iteration
>using the Zoom Bleach function over ~2um of the
>cell--this yields ~80% bleaching. For imaging,
>we are using very low laser excitation, finding
>cells with transmitted light and are not using
>the live image for image focus--typical live
>imaging stuff. For the cells that do not get
>extra bright after bleaching, the recovery rates
>are similar to published results. > > Ideas I
>have come up with are: > - Unquenching upon
>bleaching: If this were the case, I would expect
>that lowering the power of the FRAP settings
>would result in a brightening of the FRAP region
>(instead of bleaching all molecules in the
>volume, only some would bleach and create
>regional unquenching), not just reduction in
>photobleaching. > - Phototoxicity: Reducing the
>FRAP laser power or iterations or increasing
>scan speed only results in reduced bleaching, so
>I'm not sure how best to control for this.
>Perhaps adding Oxyrase or Trolox to give the
>cells some help against the ROS? > > Many
>thanks! > Wendy > > ~~~~~~~~~~~~~~~~~~~~~~~ >
>Wendy Salmon > Light Microscopy Specialist >
>Whitehead Institute for Biomedical Research >
>W.M. Keck Imaging Facility > 9 Cambridge Center,
>Rm 447 > Cambridge, MA 02142 > e:
>[log in to unmask] > w:
>http://staffa.wi.mit.edu/microscopy/"
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Box 2348), Sechelt, BC, Canada, V0N3A0,
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