acceptor photobleaching

[mmodel]

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Dear List:

As I understand it, in the acceptor photobleaching method, one monitors disappearance of the acceptor fluorescence and then measures the donor. This assumes that the donor absorption has been destroyed as well. But quantum yield and absorption are not the same, and fading of fluorescence should not necessarily indicate that there is no absorption left. Has anyone looked at it? Or am I misunderstanding something major? Thank you!

Mike

[Benjamin Smith]

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With acceptor photobleaching, the way to think about it is that the
absorption of the donor isn't changing, but rather the quantum yield (i.e.
more relaxation events go down the emission path rather than the FRET path,
as the acceptor can no longer quench donor fluorescence).  However,
depending on the donor, photobleaching of the donor can indeed be an issue,
so often when doing acceptor photobleaching, we would do it in a series of
photobleaching rounds and look at the correlation between acceptor
intensity and donor intensity during each round.  With FRET, you would
expect a perfect anticorrelation, and without FRET you would expect a
perfect correlation.  An added bonus is that this also prevents
overshooting the acceptor photobleaching, where you wind up only
photobleaching the donor.  The following paper shows how FRET was
quantified using the above method (figures 8 and S10):
http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523

If you have FLIM capability, an added control you can do is to measure the
lifetime both before and after photobleaching the acceptor.  Before
acceptor photobleaching you would see the shortened donor lifetime, and
after photobleaching you would expect to see the full donor lifetime.  The
FLIM analysis controls for a photobleaching false positive we've run into
on a confocal, where an autofluorescent structure would get brighter with
each round of photobleaching (although unlike with FRET, this would not
plateau), and the photobleaching analysis controls for a FLIM false
positive where the shortened lifetime can be just a convolution of the
lifetimes of one or more autofluorescent structures and your protein, or
purely autofluorescence (although phasor-FLIM can also help you resolve
this).

Hope this helps,
   Ben Smith


On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> *****
>
> Dear List:
>
> As I understand it, in the acceptor photobleaching method, one monitors
> disappearance of the acceptor fluorescence and then measures the donor.
> This assumes that the donor absorption has been destroyed as well. But
> quantum yield and absorption are not the same, and fading of fluorescence
> should not necessarily indicate that there is no absorption left. Has
> anyone looked at it? Or am I misunderstanding something major? Thank you!
>
> Mike
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[mmodel]

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*****

Thank you very much, Ben,


But now I see that I wrote not what I meant. The question is whether the ACCEPTOR absorption has been removed by exposure to bright light and not just its quantum yield. Because if it is only the ability of acceptor to fluoresce has been destroyed but its absorption not sufficiently reduced, then FRET will still occur. Sorry for the mistake


Mike


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Benjamin E Smith <[hidden email]>
Sent: Monday, June 25, 2018 10:37 PM
To: [hidden email]
Subject: Re: acceptor photobleaching

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Post images on http://www.imgur.com and include the link in your posting.
*****

With acceptor photobleaching, the way to think about it is that the
absorption of the donor isn't changing, but rather the quantum yield (i.e.
more relaxation events go down the emission path rather than the FRET path,
as the acceptor can no longer quench donor fluorescence).  However,
depending on the donor, photobleaching of the donor can indeed be an issue,
so often when doing acceptor photobleaching, we would do it in a series of
photobleaching rounds and look at the correlation between acceptor
intensity and donor intensity during each round.  With FRET, you would
expect a perfect anticorrelation, and without FRET you would expect a
perfect correlation.  An added bonus is that this also prevents
overshooting the acceptor photobleaching, where you wind up only
photobleaching the donor.  The following paper shows how FRET was
quantified using the above method (figures 8 and S10):
http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523

If you have FLIM capability, an added control you can do is to measure the
lifetime both before and after photobleaching the acceptor.  Before
acceptor photobleaching you would see the shortened donor lifetime, and
after photobleaching you would expect to see the full donor lifetime.  The
FLIM analysis controls for a photobleaching false positive we've run into
on a confocal, where an autofluorescent structure would get brighter with
each round of photobleaching (although unlike with FRET, this would not
plateau), and the photobleaching analysis controls for a FLIM false
positive where the shortened lifetime can be just a convolution of the
lifetimes of one or more autofluorescent structures and your protein, or
purely autofluorescence (although phasor-FLIM can also help you resolve
this).

Hope this helps,
   Ben Smith


On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> *****
>
> Dear List:
>
> As I understand it, in the acceptor photobleaching method, one monitors
> disappearance of the acceptor fluorescence and then measures the donor.
> This assumes that the donor absorption has been destroyed as well. But
> quantum yield and absorption are not the same, and fading of fluorescence
> should not necessarily indicate that there is no absorption left. Has
> anyone looked at it? Or am I misunderstanding something major? Thank you!
>
> Mike
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Benjamin Smith]

*****
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.
*****

Whether the acceptor fluorophore still has a high absorption coefficient
after photobleaching can be empirically answered with one observation: When
the acceptor is fully photobleached, the donor emission lifetime becomes
the same as free donor in solution with no acceptor present.  This suggests
that once the acceptor is photobleached, it's absorption coefficient in the
donor emission wavelength range is reduced below practically measurable
levels.  Otherwise, if the pair still underwent FRET after photobleaching,
it would have resulted in a measurable reduction in the donor's lifetime.
As for the why, irreversible photobleaching involves covalent crosslionking
to other molecules, most often oxygen.  This will completely change the
orbital structure of the acceptor fluorophore, removing the meta-stable
energy states that matched the S1 energy state of the donor.  This makes
the FRET efficiency go towards zero as J-lamda (the integral of the overlap
between the donor emission spectrum and acceptor excitation spectrum) is
effectively zero.

Cheers,
   Ben Smith

On Tue, Jun 26, 2018 at 3:24 AM, MODEL, MICHAEL <[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.
> *****
>
> Thank you very much, Ben,
>
>
> But now I see that I wrote not what I meant. The question is whether the
> ACCEPTOR absorption has been removed by exposure to bright light and not
> just its quantum yield. Because if it is only the ability of acceptor to
> fluoresce has been destroyed but its absorption not sufficiently reduced,
> then FRET will still occur. Sorry for the mistake
>
>
> Mike
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Benjamin E Smith <[hidden email]>
> Sent: Monday, June 25, 2018 10:37 PM
> To: [hidden email]
> Subject: Re: acceptor photobleaching
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.
> umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> lists.umn.edu
> [hidden email]: listserv archives. confocalmicroscopy
>
>
>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> With acceptor photobleaching, the way to think about it is that the
> absorption of the donor isn't changing, but rather the quantum yield (i.e.
> more relaxation events go down the emission path rather than the FRET path,
> as the acceptor can no longer quench donor fluorescence).  However,
> depending on the donor, photobleaching of the donor can indeed be an issue,
> so often when doing acceptor photobleaching, we would do it in a series of
> photobleaching rounds and look at the correlation between acceptor
> intensity and donor intensity during each round.  With FRET, you would
> expect a perfect anticorrelation, and without FRET you would expect a
> perfect correlation.  An added bonus is that this also prevents
> overshooting the acceptor photobleaching, where you wind up only
> photobleaching the donor.  The following paper shows how FRET was
> quantified using the above method (figures 8 and S10):
> http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523
>
> If you have FLIM capability, an added control you can do is to measure the
> lifetime both before and after photobleaching the acceptor.  Before
> acceptor photobleaching you would see the shortened donor lifetime, and
> after photobleaching you would expect to see the full donor lifetime.  The
> FLIM analysis controls for a photobleaching false positive we've run into
> on a confocal, where an autofluorescent structure would get brighter with
> each round of photobleaching (although unlike with FRET, this would not
> plateau), and the photobleaching analysis controls for a FLIM false
> positive where the shortened lifetime can be just a convolution of the
> lifetimes of one or more autofluorescent structures and your protein, or
> purely autofluorescence (although phasor-FLIM can also help you resolve
> this).
>
> Hope this helps,
>    Ben Smith
>
>
> On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> > *****
> >
> > Dear List:
> >
> > As I understand it, in the acceptor photobleaching method, one monitors
> > disappearance of the acceptor fluorescence and then measures the donor.
> > This assumes that the donor absorption has been destroyed as well. But
> > quantum yield and absorption are not the same, and fading of fluorescence
> > should not necessarily indicate that there is no absorption left. Has
> > anyone looked at it? Or am I misunderstanding something major? Thank you!
> >
> > Mike
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[mmodel]

*****
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.
*****

That would require FLIM in addition to acceptor photobleaching! Or to have enough of it to measure the absorbance.
I gather people have not been too concerned about such things when doing photobleaching experiments.
Thank you, Ben!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Benjamin E Smith
Sent: Tuesday, June 26, 2018 4:48 PM
To: [hidden email]
Subject: Re: acceptor photobleaching CORRECTION

*****
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.
*****

Whether the acceptor fluorophore still has a high absorption coefficient after photobleaching can be empirically answered with one observation: When the acceptor is fully photobleached, the donor emission lifetime becomes the same as free donor in solution with no acceptor present.  This suggests that once the acceptor is photobleached, it's absorption coefficient in the donor emission wavelength range is reduced below practically measurable levels.  Otherwise, if the pair still underwent FRET after photobleaching, it would have resulted in a measurable reduction in the donor's lifetime.
As for the why, irreversible photobleaching involves covalent crosslionking to other molecules, most often oxygen.  This will completely change the orbital structure of the acceptor fluorophore, removing the meta-stable energy states that matched the S1 energy state of the donor.  This makes the FRET efficiency go towards zero as J-lamda (the integral of the overlap between the donor emission spectrum and acceptor excitation spectrum) is effectively zero.

Cheers,
   Ben Smith

On Tue, Jun 26, 2018 at 3:24 AM, MODEL, MICHAEL <[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.
> *****
>
> Thank you very much, Ben,
>
>
> But now I see that I wrote not what I meant. The question is whether
> the ACCEPTOR absorption has been removed by exposure to bright light
> and not just its quantum yield. Because if it is only the ability of
> acceptor to fluoresce has been destroyed but its absorption not
> sufficiently reduced, then FRET will still occur. Sorry for the
> mistake
>
>
> Mike
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Benjamin E Smith <[hidden email]>
> Sent: Monday, June 25, 2018 10:37 PM
> To: [hidden email]
> Subject: Re: acceptor photobleaching
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.
> umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> lists.umn.edu
> [hidden email]: listserv archives.
> confocalmicroscopy
>
>
>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> With acceptor photobleaching, the way to think about it is that the
> absorption of the donor isn't changing, but rather the quantum yield (i.e.
> more relaxation events go down the emission path rather than the FRET
> path, as the acceptor can no longer quench donor fluorescence).  
> However, depending on the donor, photobleaching of the donor can
> indeed be an issue, so often when doing acceptor photobleaching, we
> would do it in a series of photobleaching rounds and look at the
> correlation between acceptor intensity and donor intensity during each
> round.  With FRET, you would expect a perfect anticorrelation, and
> without FRET you would expect a perfect correlation.  An added bonus
> is that this also prevents overshooting the acceptor photobleaching,
> where you wind up only photobleaching the donor.  The following paper
> shows how FRET was quantified using the above method (figures 8 and S10):
> http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523
>
> If you have FLIM capability, an added control you can do is to measure
> the lifetime both before and after photobleaching the acceptor.  
> Before acceptor photobleaching you would see the shortened donor
> lifetime, and after photobleaching you would expect to see the full
> donor lifetime.  The FLIM analysis controls for a photobleaching false
> positive we've run into on a confocal, where an autofluorescent
> structure would get brighter with each round of photobleaching
> (although unlike with FRET, this would not plateau), and the
> photobleaching analysis controls for a FLIM false positive where the
> shortened lifetime can be just a convolution of the lifetimes of one
> or more autofluorescent structures and your protein, or purely
> autofluorescence (although phasor-FLIM can also help you resolve this).
>
> Hope this helps,
>    Ben Smith
>
>
> On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> > *****
> >
> > Dear List:
> >
> > As I understand it, in the acceptor photobleaching method, one
> > monitors disappearance of the acceptor fluorescence and then measures the donor.
> > This assumes that the donor absorption has been destroyed as well.
> > But quantum yield and absorption are not the same, and fading of
> > fluorescence should not necessarily indicate that there is no
> > absorption left. Has anyone looked at it? Or am I misunderstanding something major? Thank you!
> >
> > Mike
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[mmodel]

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

For example, Fig. 1 from Bezdetnaya et al, Photochemistry and Photobiology, 1996, v 64, 382-386 shows kinetics of absorbance and fluorescence decay of hematoporphyrin under illumination. Fluorescence decays much faster.


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of MODEL, MICHAEL <[hidden email]>
Sent: Tuesday, June 26, 2018 5:17 PM
To: [hidden email]
Subject: Re: acceptor photobleaching CORRECTION

*****
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lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy



Post images on http://www.imgur.com and include the link in your posting.
*****

That would require FLIM in addition to acceptor photobleaching! Or to have enough of it to measure the absorbance.
I gather people have not been too concerned about such things when doing photobleaching experiments.
Thank you, Ben!

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Benjamin E Smith
Sent: Tuesday, June 26, 2018 4:48 PM
To: [hidden email]
Subject: Re: acceptor photobleaching CORRECTION

*****
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.
*****

Whether the acceptor fluorophore still has a high absorption coefficient after photobleaching can be empirically answered with one observation: When the acceptor is fully photobleached, the donor emission lifetime becomes the same as free donor in solution with no acceptor present.  This suggests that once the acceptor is photobleached, it's absorption coefficient in the donor emission wavelength range is reduced below practically measurable levels.  Otherwise, if the pair still underwent FRET after photobleaching, it would have resulted in a measurable reduction in the donor's lifetime.
As for the why, irreversible photobleaching involves covalent crosslionking to other molecules, most often oxygen.  This will completely change the orbital structure of the acceptor fluorophore, removing the meta-stable energy states that matched the S1 energy state of the donor.  This makes the FRET efficiency go towards zero as J-lamda (the integral of the overlap between the donor emission spectrum and acceptor excitation spectrum) is effectively zero.

Cheers,
   Ben Smith

On Tue, Jun 26, 2018 at 3:24 AM, MODEL, MICHAEL <[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.
> *****
>
> Thank you very much, Ben,
>
>
> But now I see that I wrote not what I meant. The question is whether
> the ACCEPTOR absorption has been removed by exposure to bright light
> and not just its quantum yield. Because if it is only the ability of
> acceptor to fluoresce has been destroyed but its absorption not
> sufficiently reduced, then FRET will still occur. Sorry for the
> mistake
>
>
> Mike
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Benjamin E Smith <[hidden email]>
> Sent: Monday, June 25, 2018 10:37 PM
> To: [hidden email]
> Subject: Re: acceptor photobleaching
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.
> umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> lists.umn.edu
> [hidden email]: listserv archives.
> confocalmicroscopy
>
>
>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> With acceptor photobleaching, the way to think about it is that the
> absorption of the donor isn't changing, but rather the quantum yield (i.e.
> more relaxation events go down the emission path rather than the FRET
> path, as the acceptor can no longer quench donor fluorescence).
> However, depending on the donor, photobleaching of the donor can
> indeed be an issue, so often when doing acceptor photobleaching, we
> would do it in a series of photobleaching rounds and look at the
> correlation between acceptor intensity and donor intensity during each
> round.  With FRET, you would expect a perfect anticorrelation, and
> without FRET you would expect a perfect correlation.  An added bonus
> is that this also prevents overshooting the acceptor photobleaching,
> where you wind up only photobleaching the donor.  The following paper
> shows how FRET was quantified using the above method (figures 8 and S10):
> http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523
>
> If you have FLIM capability, an added control you can do is to measure
> the lifetime both before and after photobleaching the acceptor.
> Before acceptor photobleaching you would see the shortened donor
> lifetime, and after photobleaching you would expect to see the full
> donor lifetime.  The FLIM analysis controls for a photobleaching false
> positive we've run into on a confocal, where an autofluorescent
> structure would get brighter with each round of photobleaching
> (although unlike with FRET, this would not plateau), and the
> photobleaching analysis controls for a FLIM false positive where the
> shortened lifetime can be just a convolution of the lifetimes of one
> or more autofluorescent structures and your protein, or purely
> autofluorescence (although phasor-FLIM can also help you resolve this).
>
> Hope this helps,
>    Ben Smith
>
>
> On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> > *****
> >
> > Dear List:
> >
> > As I understand it, in the acceptor photobleaching method, one
> > monitors disappearance of the acceptor fluorescence and then measures the donor.
> > This assumes that the donor absorption has been destroyed as well.
> > But quantum yield and absorption are not the same, and fading of
> > fluorescence should not necessarily indicate that there is no
> > absorption left. Has anyone looked at it? Or am I misunderstanding something major? Thank you!
> >
> > Mike
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Benjamin Smith]

*****
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.
*****

The paper addresses this, and sounds like it is a unique property of
porphyrins: "This difference reflects the difference in photosensitivity of
monomeric and aggregated species for both dyes and indicates that, under
irradiation, photobleaching of the monomers presumbly takes place. This is
consistent with the fact that the QY of fluorescence of the monomeric forms
is much higher than that of the aggregates (18). In aggregated species
internal crossing is increased and consequently fluorescence and
intersystem crossing are decreased. On the contrary, absorbance mainly
results from aggregated forms, which are photochemically stable, and to a
much lesser extent from monomeric, photolabile forms. Analysis of
difference absorption spectra of preirradiated PP IX against PP IX shows
that the wavelength dependence of the decay in absorbance closely resemble
the fluorescence excitation spectra with a discrepancy at the maximum of no
more than 2 nm (data not shown). This coincidence of spectra indicates that
only disaggregated forms of PP IX are photolabile, while aggregates are
essentially more stable."

Basically, the aggregates self-quench so they do not fluoresce, but are
still absorbent  Since they are also more photostable than the monomers,
the absorbance (aggregates and monomers) decays more slowly than the
fluorescence (monomers only).  For other dyes to have the same effect, they
too would have to form both self-quenching, photostable aggregates and
fluorescent monomers.  Otherwise, the absorbance and fluorescence would
decay more identically.

On Tue, Jun 26, 2018 at 7:03 PM, MODEL, MICHAEL <[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.
> *****
>
> For example, Fig. 1 from Bezdetnaya et al, Photochemistry and
> Photobiology, 1996, v 64, 382-386 shows kinetics of absorbance and
> fluorescence decay of hematoporphyrin under illumination. Fluorescence
> decays much faster.
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of MODEL, MICHAEL <[hidden email]>
> Sent: Tuesday, June 26, 2018 5:17 PM
> To: [hidden email]
> Subject: Re: acceptor photobleaching CORRECTION
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.
> umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> lists.umn.edu
> [hidden email]: listserv archives. confocalmicroscopy
>
>
>
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> That would require FLIM in addition to acceptor photobleaching! Or to have
> enough of it to measure the absorbance.
> I gather people have not been too concerned about such things when doing
> photobleaching experiments.
> Thank you, Ben!
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On
> Behalf Of Benjamin E Smith
> Sent: Tuesday, June 26, 2018 4:48 PM
> To: [hidden email]
> Subject: Re: acceptor photobleaching CORRECTION
>
> *****
> 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.
> *****
>
> Whether the acceptor fluorophore still has a high absorption coefficient
> after photobleaching can be empirically answered with one observation: When
> the acceptor is fully photobleached, the donor emission lifetime becomes
> the same as free donor in solution with no acceptor present.  This suggests
> that once the acceptor is photobleached, it's absorption coefficient in the
> donor emission wavelength range is reduced below practically measurable
> levels.  Otherwise, if the pair still underwent FRET after photobleaching,
> it would have resulted in a measurable reduction in the donor's lifetime.
> As for the why, irreversible photobleaching involves covalent
> crosslionking to other molecules, most often oxygen.  This will completely
> change the orbital structure of the acceptor fluorophore, removing the
> meta-stable energy states that matched the S1 energy state of the donor.
> This makes the FRET efficiency go towards zero as J-lamda (the integral of
> the overlap between the donor emission spectrum and acceptor excitation
> spectrum) is effectively zero.
>
> Cheers,
>    Ben Smith
>
> On Tue, Jun 26, 2018 at 3:24 AM, MODEL, MICHAEL <[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.
> > *****
> >
> > Thank you very much, Ben,
> >
> >
> > But now I see that I wrote not what I meant. The question is whether
> > the ACCEPTOR absorption has been removed by exposure to bright light
> > and not just its quantum yield. Because if it is only the ability of
> > acceptor to fluoresce has been destroyed but its absorption not
> > sufficiently reduced, then FRET will still occur. Sorry for the
> > mistake
> >
> >
> > Mike
> >
> >
> > ________________________________
> > From: Confocal Microscopy List <[hidden email]> on
> > behalf of Benjamin E Smith <[hidden email]>
> > Sent: Monday, June 25, 2018 10:37 PM
> > To: [hidden email]
> > Subject: Re: acceptor photobleaching
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> > LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.
> > umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > lists.umn.edu
> > [hidden email]: listserv archives.
> > confocalmicroscopy
> >
> >
> >
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > With acceptor photobleaching, the way to think about it is that the
> > absorption of the donor isn't changing, but rather the quantum yield
> (i.e.
> > more relaxation events go down the emission path rather than the FRET
> > path, as the acceptor can no longer quench donor fluorescence).
> > However, depending on the donor, photobleaching of the donor can
> > indeed be an issue, so often when doing acceptor photobleaching, we
> > would do it in a series of photobleaching rounds and look at the
> > correlation between acceptor intensity and donor intensity during each
> > round.  With FRET, you would expect a perfect anticorrelation, and
> > without FRET you would expect a perfect correlation.  An added bonus
> > is that this also prevents overshooting the acceptor photobleaching,
> > where you wind up only photobleaching the donor.  The following paper
> > shows how FRET was quantified using the above method (figures 8 and S10):
> > http://www.plantphysiol.org/content/early/2017/07/19/pp.17.00523
> >
> > If you have FLIM capability, an added control you can do is to measure
> > the lifetime both before and after photobleaching the acceptor.
> > Before acceptor photobleaching you would see the shortened donor
> > lifetime, and after photobleaching you would expect to see the full
> > donor lifetime.  The FLIM analysis controls for a photobleaching false
> > positive we've run into on a confocal, where an autofluorescent
> > structure would get brighter with each round of photobleaching
> > (although unlike with FRET, this would not plateau), and the
> > photobleaching analysis controls for a FLIM false positive where the
> > shortened lifetime can be just a convolution of the lifetimes of one
> > or more autofluorescent structures and your protein, or purely
> > autofluorescence (although phasor-FLIM can also help you resolve this).
> >
> > Hope this helps,
> >    Ben Smith
> >
> >
> > On Mon, Jun 25, 2018 at 1:39 PM, MODEL, MICHAEL <[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.
> > > *****
> > >
> > > Dear List:
> > >
> > > As I understand it, in the acceptor photobleaching method, one
> > > monitors disappearance of the acceptor fluorescence and then measures
> the donor.
> > > This assumes that the donor absorption has been destroyed as well.
> > > But quantum yield and absorption are not the same, and fading of
> > > fluorescence should not necessarily indicate that there is no
> > > absorption left. Has anyone looked at it? Or am I misunderstanding
> something major? Thank you!
> > >
> > > Mike
> > >
> >
> >
> >
> > --
> > Benjamin E. Smith, Ph. D.
> > Imaging Specialist, Vision Science
> > University of California, Berkeley
> > 195 Life Sciences Addition
> > Berkeley, CA  94720-3200
> > Tel  [phone]
> > Fax [phone]
> > e-mail: [hidden email]
> > http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>