Increase in cell brightness after FRAP

[Wendy Salmon]

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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: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/ 

[Julio Vazquez]

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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: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/ 

[Julio Vazquez]

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... maybe one way to test that is to run a time series with higher than normal laser power (and accordingly lower gain) and see if intensity increases over time...


Julio

--



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: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/ 

[John Oreopoulos]

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Julio is probably right here - sounds like de-quenching to me. Try diluting the label and repeat the experiment.

John Oreopoulos

> On Sep 19, 2014, at 5:30 PM, Julio Vazquez <[hidden email]> 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.
> *****
>
> ... maybe one way to test that is to run a time series with higher than normal laser power (and accordingly lower gain) and see if intensity increases over time...
>
>
> Julio
>
> --
>
>
>
>> 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: [hidden email]
>> w: http://staffa.wi.mit.edu/microscopy/ 

[Feinstein, Timothy]

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We saw something oddly similar when a user of my core tried
photoconverting cytoplasmic mEOS in a neurite.  The whole cell got a lot
brighter.  In that case I am not sure dequenching can explain it as the
expression level was not even all that high.  OTOH if mEOS oligomerizes
more that I thought then de-quenching makes perfect sense, as the
quenching effect would be basically independent of expression level...

All the best,


Tim

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







On 9/19/14, 8:57 PM, "John Oreopoulos" <[hidden email]> wrote:

>*****
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>roscopy
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>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gICA
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>posting.
>*****
>
>Julio is probably right here - sounds like de-quenching to me. Try
>diluting the label and repeat the experiment.
>
>John Oreopoulos
>
>> On Sep 19, 2014, at 5:30 PM, Julio Vazquez <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gIH
>>UI4N8TaQ&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>icroscopy
>> Post images on
>>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gIC
>>AJstVHOQ&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your
>>posting.
>> *****
>>
>> ... maybe one way to test that is to run a time series with higher than
>>normal laser power (and accordingly lower gain) and see if intensity
>>increases over time...
>>
>>
>> Julio
>>
>> --
>>
>>
>>
>>> On Sep 19, 2014, at 1:43 PM, Wendy Salmon wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>
>>>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gI
>>>HUI4N8TaQ&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca
>>>lmicroscopy
>>> Post images on
>>>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gI
>>>CAJstVHOQ&u=http%3a%2f%2fwww%2eimgur%2ecom 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: [hidden email]
>>> w:
>>>http://scanmail.trustwave.com/?c=129&d=sNGc1KNOulqeXQw6JwAjZ712KtYsSZ9gI
>>>HUOttETOg&u=http%3a%2f%2fstaffa%2ewi%2emit%2eedu%2fmicroscopy%2f

[Zdenek Svindrych]

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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



---------- Původní zpráva ----------
Od: Julio Vazquez <[hidden email]>
Komu: [hidden email]
Datum: 19. 9. 2014 23:29:07
Předmět: Re: Increase in cell brightness after FRAP

"*****
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*****

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: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/"

[James Pawley]

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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

>*****
>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.
>*****
>
>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
><[hidden email]> Komu:
>[hidden email] 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:
>[hidden email] > w:
>http://staffa.wi.mit.edu/microscopy/"

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Box 2348), Sechelt, BC, Canada, V0N3A0,
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Do you know which variant of GFP is being used?  Although it is unlikely to
be wtGFP, you might make sure.  Some of those constructs are still floating
around and have worked their way back into use.  The wtGFP undergoes
photoconversion/photoactivation in which the absorption at 488 increases
when irradiated with high levels if ~400nm light (this is well known) and
also with high 488nm light (not so well known).
It sounds like you are easily getting the desired bleach depth in the focal
plane of the ROI, but maybe elsewhere in the cell you are getting some
photoconversion.
Like I said it's unlikely to be wtGFP, but should be easy to rule out.
George

On Fri, Sep 19, 2014 at 4:43 PM, Wendy Salmon <[hidden email]> 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: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/
>

[Reece, Jeff (NIH/NIDDK) [E]]

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Hi Wendy,

Not ignoring all the possibilities mentioned so far, have you checked for a change in cell volume?  Perhaps it is a small chance, but you could be seeing a physiological change that is associated with the change you were hoping to monitor, so you might actually be able to do the experiment after all, after normalizing for cell volume changes (requiring a second probe for that).

One possibility to deal with some of these issues: use a fluorescent variant that bleaches more easily, so you are less likely to damage the cell in unwanted ways.  Of course you have to worry more about photobleaching, but sometimes correcting for that is easier than worrying about unwanted cell damage.  

Cheers,
Jeff

-----Original Message-----
From: James Pawley [mailto:[hidden email]]
Sent: Saturday, September 20, 2014 8:55 AM
To: [hidden email]
Subject: Re: Increase in cell brightness after FRAP

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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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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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
><[hidden email]> Komu:
>[hidden email] 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:
>[hidden email] > w:
>http://staffa.wi.mit.edu/microscopy/"

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone [phone], email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Stanislav Vitha-2]

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perhaps the culture medium plays a role?
see for instance Bogdanov, A. M., Kudryavtseva, E. I., and Lukyanov, K. A.
(2012) Anti-fading media for live cell GFP imaging, PLoS One 7, e53004.

 
I had a similar experience when doing FRAP of GFP or YFP tagged
cytoskeletal proteins in plant chloroplasts in intact leaf tissue.  Bleaching
with a 405 nm laser bleached the chlorophyll but caused a bright spot in
the green channel, or a donut-shaped spot (dark spot surrounded by
brighter area.
I attributed that to photoswitching of the fluorescent protein from the dark
state to the fluorescent state.  the darker center of the donut was where
the actual photobleaching occurred.

It seems many fluorescent proteins can be photoswitched, at least few
times.  Here is an example of mOrange2 photoswitching - cytoskeletal
protein expressed in yeast, alternating 543nm excitation (5 frames) and
405 nm (5 frames).
http://i1219.photobucket.com/albums/dd436/bohemyan/MIC/Pom_2013-
03-mOrange2-photoswitch.png

Stan Vitha
Microscopy and Imaging Center
Texas A&M Univeristy

[Wendy Salmon]

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Thank you all for your suggestions!

As many of you suggested, it looks like it is indeed quenching. The protein of interest is a known dimer so it's also possible that she got "lucky" and her GFP happens to be located in such a way it quenches when dimerized or it could be expression level. She is moving forward with plans to tease out those options.

For those of you interested in our troubleshooting strategy: Before posting to the listserv we attempted to determine if there was quenching by modulating the FRAP intensity and looking for increase of the FRAP area intensity immediately after the "bleach" event but we didn't see that--just bleaching or no bleaching. In retrospect we were probably too course with our setting choices and missed the sweat spot that would cause de-quenching. After confirmation from listserv suggestions that quenching was a likely cause, we tried a different strategy--i ncreasing the imaging illumination intensity a bit but not doing the bleaching event (ie, bleaching the whole cell). We finally saw intensity of the whole cell increase then decrease with similar rates. This is, of course, consistent with initial bleaching of the "quenching" population, thereby allowing emission, followed by bleaching of the remaining emitting population.

I am reminded again to always question your assumptions: In this case, lower intensity was not necessarily lower expression level!

Many thanks again!
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: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/ 

[John Oreopoulos]

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Dear Wendy,

Another really good, simply way to distinguish dimerazation of the fluorescent protein from photoconversion is to look at the polarization of fluorescence emission. See for example these papers:

Ghosh, S., Saha, S., Goswami, D., Bilgrami, S., and Mayor, S. (2012). Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy. In P. M. Conn (Ed.), Method enzymol. (Vol. Volume 505, pp. 291-327): Academic Press.

Tramier, M., and Coppey-Moisan, M. (2008). Fluorescence anisotropy imaging microscopy for homo-FRET in living cells Fluorescent proteins, second edition (Vol. 85, pp. 395-414). San Diego: Elsevier Academic Press Inc.

And in the case of quenching, initially the fluorescence should be highly depolarized, and as you bleach a region, it should become more polarized. It is possible to even tease out the degree of oligomerization from experiments that vary the amount of labeled vs. non-labeled protein. See this papers for example:

Squire, A., Verveer, P. J., Rocks, O., and Bastiaens, P. I. H. (2004). Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells. J. Struct. Biol., 147, 62-69.

Yeow, E. K. L., and Clayton, A. H. A. (2007). Enumeration of oligomerization states of membrane proteins in living cells by homo-FRET spectroscopy and microscopy: Theory and application. Biophys. J., 92, 3098-3104.

Adding polarization optics to a widefield fluorescence microscope is fairly straightforward, on a confocal it's a little more work but it can be done for laser scanners and spinning disks.

Cheers,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2014-09-26, at 9:55 AM, 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.
> *****
>
> Thank you all for your suggestions!
>
> As many of you suggested, it looks like it is indeed quenching. The protein of interest is a known dimer so it's also possible that she got "lucky" and her GFP happens to be located in such a way it quenches when dimerized or it could be expression level. She is moving forward with plans to tease out those options.
>
> For those of you interested in our troubleshooting strategy: Before posting to the listserv we attempted to determine if there was quenching by modulating the FRAP intensity and looking for increase of the FRAP area intensity immediately after the "bleach" event but we didn't see that--just bleaching or no bleaching. In retrospect we were probably too course with our setting choices and missed the sweat spot that would cause de-quenching. After confirmation from listserv suggestions that quenching was a likely cause, we tried a different strategy--i ncreasing the imaging illumination intensity a bit but not doing the bleaching event (ie, bleaching the whole cell). We finally saw intensity of the whole cell increase then decrease with similar rates. This is, of course, consistent with initial bleaching of the "quenching" population, thereby allowing emission, followed by bleaching of the remaining emitting population.
>
> I am reminded again to always question your assumptions: In this case, lower intensity was not necessarily lower expression level!
>
> Many thanks again!
> 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: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/