Bleaching vs. exposure time at constant dosage (revisited)

[Andrew York]

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 A few years ago, I asked for references which measure if photobleaching is
(non)linear:
http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=15352

 I'm thrilled to see a recent paper which carefully, quantitatively
measures the (non)linearity of photobleaching: dx.doi.org/10.1038/nmeth.3891

 My favorite line:
"...we expected, on the basis of 40 years of literature, that
photobleaching rates would also be linear (i.e., at twice the intensity,
the photobleaching rate doubles). Instead, we found that almost all FPs
show supralinear (‘accelerated’) photobleaching."

 This is exciting stuff! Very important for anyone who wants to maximize
signal and minimize bleaching. My favorite part is, this makes a simple,
beautiful prediction which we can easily test. If photobleaching is
noticeably supralinear, then a confocal should bleach much faster in-plane
than out-of-plane.

 Anybody in the mood to check?
1. Put any 3D sample tagged with an immobilized fluorescent protein on your
point-scanning confocal
2. Image a single 2D plane long enough to bleach it measurably
3. Take a thick 3D stack and check if you burned the 2D plane more than the
3D volume
4. Optionally repeat steps 2 and 3 and estimate the relative bleaching rates
Presumably edge effects would distort the result, so large fields of view
with uniform illumination are important.

 We (quickly, casually) looked for this effect a while ago, using mEGFP in
e coli on a spinning disk. We couldn't see any enhanced in-plane bleaching,
but there's a lot of reasons we might have missed it (for example, spinning
disk is not ideal, since the out-of-focus illumination probably has lots of
hot spots due to coherence). If anyone else tries it, I'd love to hear
their results.

*****
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*****

Hats off to Cranfill et al. for characterizing and highlighting bleaching
properties in their very nice FP evaluation article since photon budgets
are so important to so many of our experiments. For organic dyes, though,
the supralinear bleaching aspect has been reported before even for CW
illumination.

I recall getting a few raised eyebrows when I showed that for some organic
dyes the photobleaching rates are supralinear and that total detected
photons per molecule goes down as intensity goes up. This is under
conditions that I believe correctly distinguished bleaching from blinking.
The effect was reproducible by me and others, and once I looked into it, I
found prior reports of the effect for organic dyes, as well (see below). I
only tested a few dyes for this, but they all showed the effect.

Here is a little bit of the bleaching data (see Suppl. Fig. 3) in a 2012
paper of mine.
http://www.nature.com/nmeth/journal/v9/n12/full/nmeth.2214.html


Nonlinear bleaching has been reported before for organic dyes under CW
illumination. Here are a couple older examples out of many.

Eggeling et al., Analytical Chemistry, 1998, vol 70, 2651-2659,
"Photobleaching of Fluorescent Dyes Under Conditions Used for
Single-Molecule Detection: Evidence of Two-Step Photolysis".

Deschenes et al., Chemical Physics Letters, 2002, vol 365, 387-395, "Single
molecule photobleaching: increasing photon yield and survival time through
suppression of two-step photolysis".

I guess my reply is 3 years too late for Andrew's original 2013 post,
though. :)

On Wed, Jun 1, 2016 at 1:43 PM, Andrew York <
[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.
> *****
>
>  A few years ago, I asked for references which measure if photobleaching is
> (non)linear:
> http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=15352
>
>  I'm thrilled to see a recent paper which carefully, quantitatively
> measures the (non)linearity of photobleaching:
> dx.doi.org/10.1038/nmeth.3891
>
>  My favorite line:
> "...we expected, on the basis of 40 years of literature, that
> photobleaching rates would also be linear (i.e., at twice the intensity,
> the photobleaching rate doubles). Instead, we found that almost all FPs
> show supralinear (‘accelerated’) photobleaching."
>
>  This is exciting stuff! Very important for anyone who wants to maximize
> signal and minimize bleaching. My favorite part is, this makes a simple,
> beautiful prediction which we can easily test. If photobleaching is
> noticeably supralinear, then a confocal should bleach much faster in-plane
> than out-of-plane.
>
>  Anybody in the mood to check?
> 1. Put any 3D sample tagged with an immobilized fluorescent protein on your
> point-scanning confocal
> 2. Image a single 2D plane long enough to bleach it measurably
> 3. Take a thick 3D stack and check if you burned the 2D plane more than the
> 3D volume
> 4. Optionally repeat steps 2 and 3 and estimate the relative bleaching
> rates
> Presumably edge effects would distort the result, so large fields of view
> with uniform illumination are important.
>
>  We (quickly, casually) looked for this effect a while ago, using mEGFP in
> e coli on a spinning disk. We couldn't see any enhanced in-plane bleaching,
> but there's a lot of reasons we might have missed it (for example, spinning
> disk is not ideal, since the out-of-focus illumination probably has lots of
> hot spots due to coherence). If anyone else tries it, I'd love to hear
> their results.
>

--
Joshua C. Vaughan
Assistant Professor
Department of Chemistry
Box 351700
University of Washington
Seattle, WA 98195
206-543-4644

[Stoltzfus, Caleb]

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*****

A while back I worked on a project where we characterized the photobleaching rates for two-photon excitation of red fluorescent proteins. Here is a link to the resulting paper (see figure 5): http://pubs.acs.org/doi/abs/10.1021/jp502477c
It is worth noting that we found higher nonlinearity in the photobleaching rate than the excitation rate under these conditions as well.
________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Joshua Vaughan <[hidden email]>
Sent: Thursday, June 2, 2016 1:20:11 AM
To: [hidden email]
Subject: Re: Bleaching vs. exposure time at constant dosage (revisited)

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Hats off to Cranfill et al. for characterizing and highlighting bleaching
properties in their very nice FP evaluation article since photon budgets
are so important to so many of our experiments. For organic dyes, though,
the supralinear bleaching aspect has been reported before even for CW
illumination.

I recall getting a few raised eyebrows when I showed that for some organic
dyes the photobleaching rates are supralinear and that total detected
photons per molecule goes down as intensity goes up. This is under
conditions that I believe correctly distinguished bleaching from blinking.
The effect was reproducible by me and others, and once I looked into it, I
found prior reports of the effect for organic dyes, as well (see below). I
only tested a few dyes for this, but they all showed the effect.

Here is a little bit of the bleaching data (see Suppl. Fig. 3) in a 2012
paper of mine.
http://www.nature.com/nmeth/journal/v9/n12/full/nmeth.2214.html


Nonlinear bleaching has been reported before for organic dyes under CW
illumination. Here are a couple older examples out of many.

Eggeling et al., Analytical Chemistry, 1998, vol 70, 2651-2659,
"Photobleaching of Fluorescent Dyes Under Conditions Used for
Single-Molecule Detection: Evidence of Two-Step Photolysis".

Deschenes et al., Chemical Physics Letters, 2002, vol 365, 387-395, "Single
molecule photobleaching: increasing photon yield and survival time through
suppression of two-step photolysis".

I guess my reply is 3 years too late for Andrew's original 2013 post,
though. :)

On Wed, Jun 1, 2016 at 1:43 PM, Andrew York <
[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.
> *****
>
>  A few years ago, I asked for references which measure if photobleaching is
> (non)linear:
> http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=15352
>
>  I'm thrilled to see a recent paper which carefully, quantitatively
> measures the (non)linearity of photobleaching:
> dx.doi.org/10.1038/nmeth.3891
>
>  My favorite line:
> "...we expected, on the basis of 40 years of literature, that
> photobleaching rates would also be linear (i.e., at twice the intensity,
> the photobleaching rate doubles). Instead, we found that almost all FPs
> show supralinear (‘accelerated’) photobleaching."
>
>  This is exciting stuff! Very important for anyone who wants to maximize
> signal and minimize bleaching. My favorite part is, this makes a simple,
> beautiful prediction which we can easily test. If photobleaching is
> noticeably supralinear, then a confocal should bleach much faster in-plane
> than out-of-plane.
>
>  Anybody in the mood to check?
> 1. Put any 3D sample tagged with an immobilized fluorescent protein on your
> point-scanning confocal
> 2. Image a single 2D plane long enough to bleach it measurably
> 3. Take a thick 3D stack and check if you burned the 2D plane more than the
> 3D volume
> 4. Optionally repeat steps 2 and 3 and estimate the relative bleaching
> rates
> Presumably edge effects would distort the result, so large fields of view
> with uniform illumination are important.
>
>  We (quickly, casually) looked for this effect a while ago, using mEGFP in
> e coli on a spinning disk. We couldn't see any enhanced in-plane bleaching,
> but there's a lot of reasons we might have missed it (for example, spinning
> disk is not ideal, since the out-of-focus illumination probably has lots of
> hot spots due to coherence). If anyone else tries it, I'd love to hear
> their results.
>

--
Joshua C. Vaughan
Assistant Professor
Department of Chemistry
Box 351700
University of Washington
Seattle, WA 98195
206-543-4644

[Zdenek Svindrych-2]

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*****

Hi Andrew,
the reason why spinning disc is not ideal is the pinhole crosstalk. It of
course depends on the actual sample, but when I tried to bleach thick 3D
specimen (100 um nail polish with Rhodamine 6G) using FRAPPA unit, I could
hardly see it with SD, but point scanning confocal showed nice bleached
cone... It's always easier to see bright feature on a dark background than a
dark feature in a "sea of fluorecence")!
Also, to figure out more about the bleaching process, it would be
interesting to study the dynamics of it, i.e. compare normal widefield
illumination with pulsed illumination (I mean short pulses... Is anyone
using supercontinuum laser for widefield fluorescence? Or another powerful
picosecond light source?).

Best, zdenek



---------- Původní zpráva ----------
Od: Andrew York <[hidden email]>
Komu: [hidden email]
Datum: 1. 6. 2016 19:14:01
Předmět: Bleaching vs. exposure time at constant dosage (revisited)

"*****
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.
*****

A few years ago, I asked for references which measure if photobleaching is
(non)linear:
http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=15352

I'm thrilled to see a recent paper which carefully, quantitatively
measures the (non)linearity of photobleaching: dx.doi.org/10.1038/nmeth.3891

My favorite line:
"...we expected, on the basis of 40 years of literature, that
photobleaching rates would also be linear (i.e., at twice the intensity,
the photobleaching rate doubles). Instead, we found that almost all FPs
show supralinear (‘accelerated’) photobleaching."

This is exciting stuff! Very important for anyone who wants to maximize
signal and minimize bleaching. My favorite part is, this makes a simple,
beautiful prediction which we can easily test. If photobleaching is
noticeably supralinear, then a confocal should bleach much faster in-plane
than out-of-plane.

Anybody in the mood to check?
1. Put any 3D sample tagged with an immobilized fluorescent protein on your
point-scanning confocal
2. Image a single 2D plane long enough to bleach it measurably
3. Take a thick 3D stack and check if you burned the 2D plane more than the
3D volume
4. Optionally repeat steps 2 and 3 and estimate the relative bleaching rates
Presumably edge effects would distort the result, so large fields of view
with uniform illumination are important.

We (quickly, casually) looked for this effect a while ago, using mEGFP in
e coli on a spinning disk. We couldn't see any enhanced in-plane bleaching,
but there's a lot of reasons we might have missed it (for example, spinning
disk is not ideal, since the out-of-focus illumination probably has lots of
hot spots due to coherence). If anyone else tries it, I'd love to hear
their results."

[Zdenek Svindrych-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Hi Andrew,
the reason why spinning disc is not ideal is the pinhole crosstalk. It of
course depends on the actual sample, but when I tried to bleach thick 3D
specimen (100 um nail polish with Rhodamine 6G) using FRAPPA unit, I could
hardly see it with SD, but point scanning confocal showed nice bleached
cone... It's always easier to see bright feature on a dark background than a
dark feature in a "sea of fluorecence")!
But back to the subject. They really did a lot of work in that paper. But
what is disturbing (at least fom my point of view) is that the supralienar
behavior is observed in the whole range of intensities. I believe (and maybe
I'm wrong) that bleaching will be linear at low intensities. After all, it's
a quantum process, so the FP would have to remember "every photon" it
emitted (literally) and increase the probability of self-destruction with
every emission event; or the destroyed FPs (or some other photo-sensitive
molecules) accumulate and poison the remaining fluorophores (decrease their
photostability). 

If the probability of bleaching is rather high (at high intensities), the
fluorescence lifetime should be affected, too.

Also, to figure out more about the bleaching process, it would be
interesting to study the dynamics of it, i.e. compare normal widefield
illumination with pulsed illumination (I mean short pulses... Is anyone
using supercontinuum laser for widefield fluorescence? Or another powerful
picosecond light source?).

Best, zdenek




---------- Původní zpráva ----------
Od: Andrew York <[hidden email]>
Komu: [hidden email]
Datum: 1. 6. 2016 19:14:01
Předmět: Bleaching vs. exposure time at constant dosage (revisited)

"*****
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.
*****

A few years ago, I asked for references which measure if photobleaching is
(non)linear:
http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=15352

I'm thrilled to see a recent paper which carefully, quantitatively
measures the (non)linearity of photobleaching: dx.doi.org/10.1038/nmeth.3891

My favorite line:
"...we expected, on the basis of 40 years of literature, that
photobleaching rates would also be linear (i.e., at twice the intensity,
the photobleaching rate doubles). Instead, we found that almost all FPs
show supralinear (‘accelerated’) photobleaching."

This is exciting stuff! Very important for anyone who wants to maximize
signal and minimize bleaching. My favorite part is, this makes a simple,
beautiful prediction which we can easily test. If photobleaching is
noticeably supralinear, then a confocal should bleach much faster in-plane
than out-of-plane.

Anybody in the mood to check?
1. Put any 3D sample tagged with an immobilized fluorescent protein on your
point-scanning confocal
2. Image a single 2D plane long enough to bleach it measurably
3. Take a thick 3D stack and check if you burned the 2D plane more than the
3D volume
4. Optionally repeat steps 2 and 3 and estimate the relative bleaching rates
Presumably edge effects would distort the result, so large fields of view
with uniform illumination are important.

We (quickly, casually) looked for this effect a while ago, using mEGFP in
e coli on a spinning disk. We couldn't see any enhanced in-plane bleaching,
but there's a lot of reasons we might have missed it (for example, spinning
disk is not ideal, since the out-of-focus illumination probably has lots of
hot spots due to coherence). If anyone else tries it, I'd love to hear
their results."

[Tim Feinstein]

*****
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*****

Hi Zdenek,

For some reason I believed that bleaching was more a question of the
chance a fluorophore would get excited twice before it relaxed, rather
than each excitation adding some fixed probablility of failure.  The
oxygen radicals created by triplet state relaxation then covalently modify
and inactivate the fluorophore, unless antioxidants like trolox and
phenylenediamine are used.  Thus I always expected bleaching to be
non-linear.  This would explain why (in my experience) an averaged
resonant scan of roughly similar brightness still causes significantly
less bleaching.   Do I have this wrong?

Best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology





On 6/2/16, 2:38 PM, "Confocal Microscopy List on behalf of Zdenek
Svindrych" <[hidden email] on behalf of
[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.
>*****
>
>Hi Andrew,
>the reason why spinning disc is not ideal is the pinhole crosstalk. It of
>course depends on the actual sample, but when I tried to bleach thick 3D
>specimen (100 um nail polish with Rhodamine 6G) using FRAPPA unit, I
>could
>hardly see it with SD, but point scanning confocal showed nice bleached
>cone... It's always easier to see bright feature on a dark background
>than a
>dark feature in a "sea of fluorecence")!
>But back to the subject. They really did a lot of work in that paper. But
>what is disturbing (at least fom my point of view) is that the
>supralienar
>behavior is observed in the whole range of intensities. I believe (and
>maybe
>I'm wrong) that bleaching will be linear at low intensities. After all,
>it's
>a quantum process, so the FP would have to remember "every photon" it
>emitted (literally) and increase the probability of self-destruction with
>every emission event; or the destroyed FPs (or some other photo-sensitive
>molecules) accumulate and poison the remaining fluorophores (decrease
>their
>photostability).
>
>If the probability of bleaching is rather high (at high intensities), the
>fluorescence lifetime should be affected, too.
>
>Also, to figure out more about the bleaching process, it would be
>interesting to study the dynamics of it, i.e. compare normal widefield
>illumination with pulsed illumination (I mean short pulses... Is anyone
>using supercontinuum laser for widefield fluorescence? Or another
>powerful
>picosecond light source?).
>
>Best, zdenek
>
>
>
>
>---------- Původní zpráva ----------
>Od: Andrew York <[hidden email]>
>Komu: [hidden email]
>Datum: 1. 6. 2016 19:14:01
>Předmět: Bleaching vs. exposure time at constant dosage (revisited)
>
>"*****
>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.
>*****
>
>A few years ago, I asked for references which measure if photobleaching is
>(non)linear:
>http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=1535
>2
>
>I'm thrilled to see a recent paper which carefully, quantitatively
>measures the (non)linearity of photobleaching:
>dx.doi.org/10.1038/nmeth.3891
>
>My favorite line:
>"...we expected, on the basis of 40 years of literature, that
>photobleaching rates would also be linear (i.e., at twice the intensity,
>the photobleaching rate doubles). Instead, we found that almost all FPs
>show supralinear ('accelerated') photobleaching."
>
>This is exciting stuff! Very important for anyone who wants to maximize
>signal and minimize bleaching. My favorite part is, this makes a simple,
>beautiful prediction which we can easily test. If photobleaching is
>noticeably supralinear, then a confocal should bleach much faster in-plane
>than out-of-plane.
>
>Anybody in the mood to check?
>1. Put any 3D sample tagged with an immobilized fluorescent protein on
>your
>point-scanning confocal
>2. Image a single 2D plane long enough to bleach it measurably
>3. Take a thick 3D stack and check if you burned the 2D plane more than
>the
>3D volume
>4. Optionally repeat steps 2 and 3 and estimate the relative bleaching
>rates
>Presumably edge effects would distort the result, so large fields of view
>with uniform illumination are important.
>
>We (quickly, casually) looked for this effect a while ago, using mEGFP in
>e coli on a spinning disk. We couldn't see any enhanced in-plane
>bleaching,
>but there's a lot of reasons we might have missed it (for example,
>spinning
>disk is not ideal, since the out-of-focus illumination probably has lots
>of
>hot spots due to coherence). If anyone else tries it, I'd love to hear
>their results."

[Kurt Thorn]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

On 6/2/2016 12:25 PM, Feinstein, Timothy N 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.
> *****
>
> Hi Zdenek,
>
> For some reason I believed that bleaching was more a question of the
> chance a fluorophore would get excited twice before it relaxed, rather
> than each excitation adding some fixed probablility of failure.  The
> oxygen radicals created by triplet state relaxation then covalently modify
> and inactivate the fluorophore, unless antioxidants like trolox and
> phenylenediamine are used.  Thus I always expected bleaching to be
> non-linear.  This would explain why (in my experience) an averaged
> resonant scan of roughly similar brightness still causes significantly
> less bleaching.   Do I have this wrong?
>
> Best,
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology

The simplest model for photobleaching is generally that with each
excitation, the protein has a constant chance of crossing into the
triplet state, interacting with oxygen, and destroying itself. This
model gives single exponential kinetics and a linear dependence of
bleaching rate on intensity. Prior to this paper, I assumed that
nonlinearity would only result when the excitation power was such that
the fluorophore could absorb a second photon when it was already in its
excited state, which you would only expect to see at high laser powers
since the fluorescence lifetimes are generally short.

However, it is also clear that photobleaching in fluorescent proteins is
more complicated than the simple model predicts, because most
fluorescent proteins do not show single exponential photobleaching. See
for instance,
http://www.nature.com/nmeth/journal/v5/n6/fig_tab/nmeth.1209_F1.html.
Given the complex kinetics of bleaching, it seems likely that there are
multiple mechanisms of bleaching for any given protein.  The Cranfill
paper doesn't report bleaching rate constants but rather time to bleach
to half intensity at a given laser power, which is going to be a
composite of these multiple rate constants.  Given that, I think it's
hard to interpret this supralinear response in terms of a mechanistic
model, but its clear that this will have important consequences for how
we do our imaging.

Also regarding the protein memory aspect, I think it's not hard to
imagine that there are conformational changes, or even chemical changes,
resulting from excitation that could have long-lived effects on the
bleaching behavior of proteins. After all, we have photoswitchable
proteins whose fluorescent properties change dramatically after
absorbing a single photon. It's not hard for me to imagine that
something similar could underlie the photobleaching behavior reported here.

Finally, I think this is a tremendous paper and I'm really glad to see
it published. I've wanted to have this sort of data available for a long
time - a comprehensive comparison of fluorescent protein data taken
under identical conditions.  They also provide data for all of the
proteins they've characterized in the supplemental material, and
detailed protocols for all their measurements.

Kurt

--
Kurt Thorn
Associate Professor
Director, Nikon Imaging Center
http://thornlab.ucsf.edu/
http://nic.ucsf.edu/blog/

[Michael Giacomelli]

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*****

Hi Tim,

That was my understanding as well.

I know transient absorption spectroscopy has been used to study light
absorption in chlorophylls by pulsing them with sequential ultrafast
pulses to map out the lifetime and spectra of excited states. Probably
something similar could be applied to any other fluorophore as well
and determine which states can absorb that second photon, how long
they last, etc and then map out the various paths to photobleaching
individual molecules.  However, I'm not sure that would be very useful
for confocal applications, it is probably enough to just know the
overall coefficient of nonlinearity so that one can compute the
advantage of linescan, spinning disk, lightsheet, etc over confocal or
2 photon.

I'd also be interested to see similar data for small molecules.
Intuitively I'd expect that a very small molecule like rhodamine or
acridine would have a lot fewer possible excited states, but I don't
know if that translates into more linear photobleaching.

Michael

On Thu, Jun 2, 2016 at 3:25 PM, Feinstein, Timothy N <[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.
> *****
>
> Hi Zdenek,
>
> For some reason I believed that bleaching was more a question of the
> chance a fluorophore would get excited twice before it relaxed, rather
> than each excitation adding some fixed probablility of failure.  The
> oxygen radicals created by triplet state relaxation then covalently modify
> and inactivate the fluorophore, unless antioxidants like trolox and
> phenylenediamine are used.  Thus I always expected bleaching to be
> non-linear.  This would explain why (in my experience) an averaged
> resonant scan of roughly similar brightness still causes significantly
> less bleaching.   Do I have this wrong?
>
> Best,
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
>
>
>
>
>
> On 6/2/16, 2:38 PM, "Confocal Microscopy List on behalf of Zdenek
> Svindrych" <[hidden email] on behalf of
> [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.
>>*****
>>
>>Hi Andrew,
>>the reason why spinning disc is not ideal is the pinhole crosstalk. It of
>>course depends on the actual sample, but when I tried to bleach thick 3D
>>specimen (100 um nail polish with Rhodamine 6G) using FRAPPA unit, I
>>could
>>hardly see it with SD, but point scanning confocal showed nice bleached
>>cone... It's always easier to see bright feature on a dark background
>>than a
>>dark feature in a "sea of fluorecence")!
>>But back to the subject. They really did a lot of work in that paper. But
>>what is disturbing (at least fom my point of view) is that the
>>supralienar
>>behavior is observed in the whole range of intensities. I believe (and
>>maybe
>>I'm wrong) that bleaching will be linear at low intensities. After all,
>>it's
>>a quantum process, so the FP would have to remember "every photon" it
>>emitted (literally) and increase the probability of self-destruction with
>>every emission event; or the destroyed FPs (or some other photo-sensitive
>>molecules) accumulate and poison the remaining fluorophores (decrease
>>their
>>photostability).
>>
>>If the probability of bleaching is rather high (at high intensities), the
>>fluorescence lifetime should be affected, too.
>>
>>Also, to figure out more about the bleaching process, it would be
>>interesting to study the dynamics of it, i.e. compare normal widefield
>>illumination with pulsed illumination (I mean short pulses... Is anyone
>>using supercontinuum laser for widefield fluorescence? Or another
>>powerful
>>picosecond light source?).
>>
>>Best, zdenek
>>
>>
>>
>>
>>---------- Původní zpráva ----------
>>Od: Andrew York <[hidden email]>
>>Komu: [hidden email]
>>Datum: 1. 6. 2016 19:14:01
>>Předmět: Bleaching vs. exposure time at constant dosage (revisited)
>>
>>"*****
>>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.
>>*****
>>
>>A few years ago, I asked for references which measure if photobleaching is
>>(non)linear:
>>http://lists.umn.edu/cgi-bin/wa?A2=ind1306&L=confocalmicroscopy&D=0&P=1535
>>2
>>
>>I'm thrilled to see a recent paper which carefully, quantitatively
>>measures the (non)linearity of photobleaching:
>>dx.doi.org/10.1038/nmeth.3891
>>
>>My favorite line:
>>"...we expected, on the basis of 40 years of literature, that
>>photobleaching rates would also be linear (i.e., at twice the intensity,
>>the photobleaching rate doubles). Instead, we found that almost all FPs
>>show supralinear ('accelerated') photobleaching."
>>
>>This is exciting stuff! Very important for anyone who wants to maximize
>>signal and minimize bleaching. My favorite part is, this makes a simple,
>>beautiful prediction which we can easily test. If photobleaching is
>>noticeably supralinear, then a confocal should bleach much faster in-plane
>>than out-of-plane.
>>
>>Anybody in the mood to check?
>>1. Put any 3D sample tagged with an immobilized fluorescent protein on
>>your
>>point-scanning confocal
>>2. Image a single 2D plane long enough to bleach it measurably
>>3. Take a thick 3D stack and check if you burned the 2D plane more than
>>the
>>3D volume
>>4. Optionally repeat steps 2 and 3 and estimate the relative bleaching
>>rates
>>Presumably edge effects would distort the result, so large fields of view
>>with uniform illumination are important.
>>
>>We (quickly, casually) looked for this effect a while ago, using mEGFP in
>>e coli on a spinning disk. We couldn't see any enhanced in-plane
>>bleaching,
>>but there's a lot of reasons we might have missed it (for example,
>>spinning
>>disk is not ideal, since the out-of-focus illumination probably has lots
>>of
>>hot spots due to coherence). If anyone else tries it, I'd love to hear
>>their results."