Photobleaching Standard

[Neil Anthony]

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Hi all, I hope the science is treating you well.

I was wondering if anybody has heard of a good standard sample for
comparing photobleaching.  I want to compare how different microscopes
perform under different conditions and wonder how much the sample plays
a role in quantifying the photobleaching rate.  I would presumably
calculate the intensity decay over a time lapse as a metric, but wasn't
sure if the sample type was a factor as I don't know much about the
photochemistry.  On that note, can anybody recommend a good reference
that discusses the subtleties of photobleaching?

Thanks in advance for your time.

Neil

[Michael Giacomelli]

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>   I would presumably calculate the intensity decay over a time lapse as a metric, but wasn't sure if the sample type was a factor as I don't know much about the photochemistry.

That is how publications I've seen do it.  Fluorophore stability also
depends on availability of reactive oxygen, temperature, etc, but
assuming your sample is consistent between systems and that your
conditions are realistic for how you will actually use the
fluorophore, that probably won't matter.

I'm assuming your intention is to compare very different types of
microscope (e.g. widefield vs. nonlinear)?  Otherwise you can probably
estimate the relative rates just from the microscope specifications.

Mike

On Wed, Mar 11, 2015 at 2:38 PM, Neil Anthony <[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 all, I hope the science is treating you well.
>
> I was wondering if anybody has heard of a good standard sample for comparing
> photobleaching.  I want to compare how different microscopes perform under
> different conditions and wonder how much the sample plays a role in
> quantifying the photobleaching rate.  I would presumably calculate the
> intensity decay over a time lapse as a metric, but wasn't sure if the sample
> type was a factor as I don't know much about the photochemistry.  On that
> note, can anybody recommend a good reference that discusses the subtleties
> of photobleaching?
>
> Thanks in advance for your time.
>
> Neil

[samjlord]

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Hi Neil,

Great question.

On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
wrote:
>I would presumably
>calculate the intensity decay over a time lapse as a metric, but wasn't
>sure if the sample type was a factor as I don't know much about the
>photochemistry.

I have a pet peeve: when a paper reports photobleaching half-lives or bleaching
decay times *without accounting for the rate of absorbing photons*.
Photobleaching is a statistical process that typically occurs from the excited state
of the molecule (e.g. from intersystem crossing to a triplet state and then further
excitation and photodegratation) or from an excited producing singlet oxygen that
then reacts with that molecule or a nearby molecule.

Either way, the rate of photobleaching is going to depend on how frequently the
dye cycles into the excited state. If you compare two samples without taking into
account the excitation rate, you might think one sample is more susceptible to
bleaching when really it is just brighter.

I recommend using the photobleaching quantum yield as an absolute measure. It
is the probability of photobleaching with each photon absorbed, or the ratio of the
bleaching rate to the photon absorption rate. It is easy to calculate if you know the
extinction coefficient of the dye and the irradiance and wavelength of light you're
using. See equation 2.1 of my paper here:

Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E. (2010).
Molecules and methods for super-resolution imaging. Methods in Enzymology,
475(10), 27–59. doi:10.1016/S0076-6879(10)75002-3
http://everydayscientist.com/pdfs/sjl16.pdf

Alternatively, maybe a more practical metric would be to change the illumination
so that each sample has the same brightness on the camera or the same signal to
noise ratio or some other useful quantity. Then record the bleaching half-time. That
would tell you exactly what you really want to know: given a certain starting image
quality, how long can I observe my sample? This measure should indeed scale with
the photobleaching quantum yield, but it also takes into account the "brightness"
of the dye (i.e. the fluorescence quantum yield times the extinction coefficient), as
well as microscope factors like the chromatic aberrations and camera quantum
efficiency at different wavelengths.

This is a pragmatic measure, but it would be a little hard to directly compare
completely different dyes, samples, microscopes, etc. That’s why I like absolute
metrics, like the photobleaching quantum yield and dye brightness.

For a great reference on dye brightness, see:

A guide to choosing fluorescent proteins
http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
s%20-%20Choosing%20fluorescent%20proteins.pdf

(Note the measure that Shaner used for photo stability does indeed take into
account absorption rate: “Time for bleaching from an initial emission rate of 1,000
photons/s down to 500 photons/s (t1/2; for comparison, fluorescein at pH 8.4 has
t1/2 of 5.2 s).”)

The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid the pitfalls of
multi-exponential fitting and reporting weighted decay constants (see Lakowicz or
equation 2.2 of my paper above). Half-life is a super simple and pragmatic
measure of sample photobleaching.

>On that note, can anybody recommend a good reference
>that discusses the subtleties of photobleaching?

As far as I know, there is no single review that really encompasses all the
subtleties of photobleaching. A lot of those subtleties are not well understood.
People often blame singlet oxygen, but it’s way more complicated than that (e.g.
triplet/ground-state oxygen is an excellent triplet quencher, so it is actually good to
have around to reduce dyes in the triplet bottleneck; see Hubner. J. Chem. Phys.
2001, 115, 9619). There are many paths to photobleaching, they are difficult to
observe, they yield a huge range of photoproducts, and each dye is likely to have a
completely different way it prefers to die.

There are some great examples of papers where the authors discovered the
photobleaching path of a particular dye or subset of dyes:

Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
Photoswitching mechanism of cyanine dyes. Journal of the American Chemical
Society, 131, 18192–3.

Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking and
switching behaviour of single molecules of green fluorescent protein. Nature, 388,
355–8.

Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with improved
photostability. Organic Letters, 9, 2775–7.

Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways in single-
molecule FRET experiments. Journal of the American Chemical Society, 129, 4643–
54.

Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of Trolox as
antiblinking and antibleaching reagent. Journal of the American Chemical Society,
131, 5018–9.

Berglund AJ. (2004). Nonexponential statistics of fluorescence photobleaching. The
Journal of Chemical Physics, 121, 2899–903.

Best,

-Sam

[Guy Cox-2]

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I've never tried to do this, but could one not use standard volumes of a standard dye concentration, for example in a multiwell slide?

                                                                                     Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sam Lord
Sent: Friday, 13 March 2015 5:41 AM
To: [hidden email]
Subject: Re: Photobleaching Standard

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Hi Neil,

Great question.

On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
wrote:
>I would presumably
>calculate the intensity decay over a time lapse as a metric, but wasn't
>sure if the sample type was a factor as I don't know much about the
>photochemistry.

I have a pet peeve: when a paper reports photobleaching half-lives or bleaching decay times *without accounting for the rate of absorbing photons*.
Photobleaching is a statistical process that typically occurs from the excited state of the molecule (e.g. from intersystem crossing to a triplet state and then further excitation and photodegratation) or from an excited producing singlet oxygen that then reacts with that molecule or a nearby molecule.

Either way, the rate of photobleaching is going to depend on how frequently the dye cycles into the excited state. If you compare two samples without taking into account the excitation rate, you might think one sample is more susceptible to bleaching when really it is just brighter.

I recommend using the photobleaching quantum yield as an absolute measure. It is the probability of photobleaching with each photon absorbed, or the ratio of the bleaching rate to the photon absorption rate. It is easy to calculate if you know the extinction coefficient of the dye and the irradiance and wavelength of light you're using. See equation 2.1 of my paper here:

Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E. (2010).
Molecules and methods for super-resolution imaging. Methods in Enzymology, 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3 http://everydayscientist.com/pdfs/sjl16.pdf

Alternatively, maybe a more practical metric would be to change the illumination so that each sample has the same brightness on the camera or the same signal to noise ratio or some other useful quantity. Then record the bleaching half-time. That would tell you exactly what you really want to know: given a certain starting image quality, how long can I observe my sample? This measure should indeed scale with the photobleaching quantum yield, but it also takes into account the "brightness"
of the dye (i.e. the fluorescence quantum yield times the extinction coefficient), as well as microscope factors like the chromatic aberrations and camera quantum efficiency at different wavelengths.

This is a pragmatic measure, but it would be a little hard to directly compare completely different dyes, samples, microscopes, etc. That's why I like absolute metrics, like the photobleaching quantum yield and dye brightness.

For a great reference on dye brightness, see:

A guide to choosing fluorescent proteins http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
s%20-%20Choosing%20fluorescent%20proteins.pdf

(Note the measure that Shaner used for photo stability does indeed take into account absorption rate: "Time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons/s (t1/2; for comparison, fluorescein at pH 8.4 has
t1/2 of 5.2 s).")

The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid the pitfalls of multi-exponential fitting and reporting weighted decay constants (see Lakowicz or equation 2.2 of my paper above). Half-life is a super simple and pragmatic measure of sample photobleaching.

>On that note, can anybody recommend a good reference that discusses the
>subtleties of photobleaching?

As far as I know, there is no single review that really encompasses all the subtleties of photobleaching. A lot of those subtleties are not well understood.
People often blame singlet oxygen, but it's way more complicated than that (e.g.
triplet/ground-state oxygen is an excellent triplet quencher, so it is actually good to have around to reduce dyes in the triplet bottleneck; see Hubner. J. Chem. Phys.
2001, 115, 9619). There are many paths to photobleaching, they are difficult to observe, they yield a huge range of photoproducts, and each dye is likely to have a completely different way it prefers to die.

There are some great examples of papers where the authors discovered the photobleaching path of a particular dye or subset of dyes:

Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
Photoswitching mechanism of cyanine dyes. Journal of the American Chemical Society, 131, 18192-3.

Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature, 388, 355-8.

Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with improved photostability. Organic Letters, 9, 2775-7.

Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways in single- molecule FRET experiments. Journal of the American Chemical Society, 129, 4643- 54.

Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of Trolox as antiblinking and antibleaching reagent. Journal of the American Chemical Society, 131, 5018-9.

Berglund AJ. (2004). Nonexponential statistics of fluorescence photobleaching. The Journal of Chemical Physics, 121, 2899-903.

Best,

-Sam

[samjlord]

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Hi Guy,

On Fri, 13 Mar 2015 13:10:05 +0000, Guy Cox <[hidden email]>
wrote:
>I've never tried to do this, but could one not use standard volumes of a standard
>dye concentration, for example in a multiwell slide?

I like that approach, but I think that the diffusion of the dye in a liquid combined
with the relatively small imaging area will mean this will take a *long* time to
bleach. And some dyes have different properties when actually bound to a protein
or antibody.

I've measured photobleaching of dyes by embedding them in agarose or gelatin
and sandwiched that between a coverslip and slide. You could add spacer
microbeads to the mixture if you want to maintain a certain thickness. If you
normalize by brightness/absorption, the exact concentration of the dye shouldn't
matter, but keep it relatively low and as similar sample-to-sample to avoid issues
such as self-quenching, dimerization, reabsorption, etc.

You could also consider something immobile like a simple immunofluorescence
sample. Of course, you'd have to remake that often, and if your staining protocol
isn't well worked out, you might get significantly different brightnesses between
samples. But it might be a starting place.

If you're just trying to compare microscope systems and not dyes, there's always
Tetraspeck beads.

[Neil Anthony]

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*****

Cool, thanks Sam.  That's exactly the kind of answer I was looking for;
it's great to get the very specific details from an expert.

Thanks
Neil



On 3/12/2015 2:40 PM, Sam Lord 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 Neil,
>
> Great question.
>
> On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
> wrote:
>> I would presumably
>> calculate the intensity decay over a time lapse as a metric, but wasn't
>> sure if the sample type was a factor as I don't know much about the
>> photochemistry.
> I have a pet peeve: when a paper reports photobleaching half-lives or bleaching
> decay times *without accounting for the rate of absorbing photons*.
> Photobleaching is a statistical process that typically occurs from the excited state
> of the molecule (e.g. from intersystem crossing to a triplet state and then further
> excitation and photodegratation) or from an excited producing singlet oxygen that
> then reacts with that molecule or a nearby molecule.
>
> Either way, the rate of photobleaching is going to depend on how frequently the
> dye cycles into the excited state. If you compare two samples without taking into
> account the excitation rate, you might think one sample is more susceptible to
> bleaching when really it is just brighter.
>
> I recommend using the photobleaching quantum yield as an absolute measure. It
> is the probability of photobleaching with each photon absorbed, or the ratio of the
> bleaching rate to the photon absorption rate. It is easy to calculate if you know the
> extinction coefficient of the dye and the irradiance and wavelength of light you're
> using. See equation 2.1 of my paper here:
>
> Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E. (2010).
> Molecules and methods for super-resolution imaging. Methods in Enzymology,
> 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3
> http://everydayscientist.com/pdfs/sjl16.pdf
>
> Alternatively, maybe a more practical metric would be to change the illumination
> so that each sample has the same brightness on the camera or the same signal to
> noise ratio or some other useful quantity. Then record the bleaching half-time. That
> would tell you exactly what you really want to know: given a certain starting image
> quality, how long can I observe my sample? This measure should indeed scale with
> the photobleaching quantum yield, but it also takes into account the "brightness"
> of the dye (i.e. the fluorescence quantum yield times the extinction coefficient), as
> well as microscope factors like the chromatic aberrations and camera quantum
> efficiency at different wavelengths.
>
> This is a pragmatic measure, but it would be a little hard to directly compare
> completely different dyes, samples, microscopes, etc. That's why I like absolute
> metrics, like the photobleaching quantum yield and dye brightness.
>
> For a great reference on dye brightness, see:
>
> A guide to choosing fluorescent proteins
> http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
> s%20-%20Choosing%20fluorescent%20proteins.pdf
>
> (Note the measure that Shaner used for photo stability does indeed take into
> account absorption rate: "Time for bleaching from an initial emission rate of 1,000
> photons/s down to 500 photons/s (t1/2; for comparison, fluorescein at pH 8.4 has
> t1/2 of 5.2 s).")
>
> The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid the pitfalls of
> multi-exponential fitting and reporting weighted decay constants (see Lakowicz or
> equation 2.2 of my paper above). Half-life is a super simple and pragmatic
> measure of sample photobleaching.
>
>> On that note, can anybody recommend a good reference
>> that discusses the subtleties of photobleaching?
> As far as I know, there is no single review that really encompasses all the
> subtleties of photobleaching. A lot of those subtleties are not well understood.
> People often blame singlet oxygen, but it's way more complicated than that (e.g.
> triplet/ground-state oxygen is an excellent triplet quencher, so it is actually good to
> have around to reduce dyes in the triplet bottleneck; see Hubner. J. Chem. Phys.
> 2001, 115, 9619). There are many paths to photobleaching, they are difficult to
> observe, they yield a huge range of photoproducts, and each dye is likely to have a
> completely different way it prefers to die.
>
> There are some great examples of papers where the authors discovered the
> photobleaching path of a particular dye or subset of dyes:
>
> Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
> Photoswitching mechanism of cyanine dyes. Journal of the American Chemical
> Society, 131, 18192-3.
>
> Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking and
> switching behaviour of single molecules of green fluorescent protein. Nature, 388,
> 355-8.
>
> Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with improved
> photostability. Organic Letters, 9, 2775-7.
>
> Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways in single-
> molecule FRET experiments. Journal of the American Chemical Society, 129, 4643-
> 54.
>
> Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of Trolox as
> antiblinking and antibleaching reagent. Journal of the American Chemical Society,
> 131, 5018-9.
>
> Berglund AJ. (2004). Nonexponential statistics of fluorescence photobleaching. The
> Journal of Chemical Physics, 121, 2899-903.
>
> Best,
>
> -Sam

[Andrew York]

*****
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*****

A reasonable standardized sample to try:
http://www.gattaquant.com/products/gatta-brightness.html
I haven't tried them myself, but I'd like to. If anyone has, please tell me
about your experience.

This wouldn't directly tell you photobleaching rates, but since you
actually know how many dye molecules you're starting with, it should make
bleaching statistics simple to gather, and simple to interpret. It won't
get at interesting sample-dependent bleaching questions, but it should help
if you want to compare microscopes quantitatively.

On Mon, Mar 16, 2015 at 1:26 PM, Neil Anthony <[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.
> *****
>
> Cool, thanks Sam.  That's exactly the kind of answer I was looking for;
> it's great to get the very specific details from an expert.
>
> Thanks
> Neil
>
>
>
>
> On 3/12/2015 2:40 PM, Sam Lord 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 Neil,
>>
>> Great question.
>>
>> On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
>> wrote:
>>
>>> I would presumably
>>> calculate the intensity decay over a time lapse as a metric, but wasn't
>>> sure if the sample type was a factor as I don't know much about the
>>> photochemistry.
>>>
>> I have a pet peeve: when a paper reports photobleaching half-lives or
>> bleaching
>> decay times *without accounting for the rate of absorbing photons*.
>> Photobleaching is a statistical process that typically occurs from the
>> excited state
>> of the molecule (e.g. from intersystem crossing to a triplet state and
>> then further
>> excitation and photodegratation) or from an excited producing singlet
>> oxygen that
>> then reacts with that molecule or a nearby molecule.
>>
>> Either way, the rate of photobleaching is going to depend on how
>> frequently the
>> dye cycles into the excited state. If you compare two samples without
>> taking into
>> account the excitation rate, you might think one sample is more
>> susceptible to
>> bleaching when really it is just brighter.
>>
>> I recommend using the photobleaching quantum yield as an absolute
>> measure. It
>> is the probability of photobleaching with each photon absorbed, or the
>> ratio of the
>> bleaching rate to the photon absorption rate. It is easy to calculate if
>> you know the
>> extinction coefficient of the dye and the irradiance and wavelength of
>> light you're
>> using. See equation 2.1 of my paper here:
>>
>> Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E.
>> (2010).
>> Molecules and methods for super-resolution imaging. Methods in Enzymology,
>> 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3
>>
>> http://everydayscientist.com/pdfs/sjl16.pdf
>>
>> Alternatively, maybe a more practical metric would be to change the
>> illumination
>> so that each sample has the same brightness on the camera or the same
>> signal to
>> noise ratio or some other useful quantity. Then record the bleaching
>> half-time. That
>> would tell you exactly what you really want to know: given a certain
>> starting image
>> quality, how long can I observe my sample? This measure should indeed
>> scale with
>> the photobleaching quantum yield, but it also takes into account the
>> "brightness"
>> of the dye (i.e. the fluorescence quantum yield times the extinction
>> coefficient), as
>> well as microscope factors like the chromatic aberrations and camera
>> quantum
>> efficiency at different wavelengths.
>>
>> This is a pragmatic measure, but it would be a little hard to directly
>> compare
>> completely different dyes, samples, microscopes, etc. That's why I like
>> absolute
>> metrics, like the photobleaching quantum yield and dye brightness.
>>
>> For a great reference on dye brightness, see:
>>
>> A guide to choosing fluorescent proteins
>> http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
>> s%20-%20Choosing%20fluorescent%20proteins.pdf
>>
>> (Note the measure that Shaner used for photo stability does indeed take
>> into
>> account absorption rate: "Time for bleaching from an initial emission
>> rate of 1,000
>> photons/s down to 500 photons/s (t1/2; for comparison, fluorescein at pH
>> 8.4 has
>> t1/2 of 5.2 s).")
>>
>> The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid the
>> pitfalls of
>> multi-exponential fitting and reporting weighted decay constants (see
>> Lakowicz or
>> equation 2.2 of my paper above). Half-life is a super simple and pragmatic
>> measure of sample photobleaching.
>>
>>  On that note, can anybody recommend a good reference
>>> that discusses the subtleties of photobleaching?
>>>
>> As far as I know, there is no single review that really encompasses all
>> the
>> subtleties of photobleaching. A lot of those subtleties are not well
>> understood.
>> People often blame singlet oxygen, but it's way more complicated than
>> that (e.g.
>> triplet/ground-state oxygen is an excellent triplet quencher, so it is
>> actually good to
>> have around to reduce dyes in the triplet bottleneck; see Hubner. J.
>> Chem. Phys.
>> 2001, 115, 9619). There are many paths to photobleaching, they are
>> difficult to
>> observe, they yield a huge range of photoproducts, and each dye is likely
>> to have a
>> completely different way it prefers to die.
>>
>> There are some great examples of papers where the authors discovered the
>> photobleaching path of a particular dye or subset of dyes:
>>
>> Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
>> Photoswitching mechanism of cyanine dyes. Journal of the American Chemical
>> Society, 131, 18192-3.
>>
>> Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking and
>> switching behaviour of single molecules of green fluorescent protein.
>> Nature, 388,
>> 355-8.
>>
>> Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with improved
>> photostability. Organic Letters, 9, 2775-7.
>>
>> Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways in
>> single-
>> molecule FRET experiments. Journal of the American Chemical Society, 129,
>> 4643-
>> 54.
>>
>> Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of Trolox as
>> antiblinking and antibleaching reagent. Journal of the American Chemical
>> Society,
>> 131, 5018-9.
>>
>> Berglund AJ. (2004). Nonexponential statistics of fluorescence
>> photobleaching. The
>> Journal of Chemical Physics, 121, 2899-903.
>>
>> Best,
>>
>> -Sam
>>
>

[mcammer]

Am I missing something?

I don’t understand how you can have a standard independent of the actual experimental condition since each fluorophore behaves differently and the specific biological/chemical environment may make a difference too.

Also, you need to measure the illumination at the sample independently from the intensity on the detection side.

Regards,
Michael

=========================================================================
 Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
                          Cell:  914-309-3270     Temporary location:  SK2-7
          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew York
Sent: Tuesday, March 17, 2015 4:36 PM
To: [hidden email]
Subject: Re: Photobleaching Standard

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A reasonable standardized sample to try:
http://www.gattaquant.com/products/gatta-brightness.html
I haven't tried them myself, but I'd like to. If anyone has, please tell me about your experience.

This wouldn't directly tell you photobleaching rates, but since you actually know how many dye molecules you're starting with, it should make bleaching statistics simple to gather, and simple to interpret. It won't get at interesting sample-dependent bleaching questions, but it should help if you want to compare microscopes quantitatively.

On Mon, Mar 16, 2015 at 1:26 PM, Neil Anthony <[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.
> *****
>
> Cool, thanks Sam.  That's exactly the kind of answer I was looking
> for; it's great to get the very specific details from an expert.
>
> Thanks
> Neil
>
>
>
>
> On 3/12/2015 2:40 PM, Sam Lord 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 Neil,
>>
>> Great question.
>>
>> On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
>> wrote:
>>
>>> I would presumably
>>> calculate the intensity decay over a time lapse as a metric, but
>>> wasn't sure if the sample type was a factor as I don't know much
>>> about the photochemistry.
>>>
>> I have a pet peeve: when a paper reports photobleaching half-lives or
>> bleaching decay times *without accounting for the rate of absorbing
>> photons*.
>> Photobleaching is a statistical process that typically occurs from
>> the excited state of the molecule (e.g. from intersystem crossing to
>> a triplet state and then further excitation and photodegratation) or
>> from an excited producing singlet oxygen that then reacts with that
>> molecule or a nearby molecule.
>>
>> Either way, the rate of photobleaching is going to depend on how
>> frequently the dye cycles into the excited state. If you compare two
>> samples without taking into account the excitation rate, you might
>> think one sample is more susceptible to bleaching when really it is
>> just brighter.
>>
>> I recommend using the photobleaching quantum yield as an absolute
>> measure. It is the probability of photobleaching with each photon
>> absorbed, or the ratio of the bleaching rate to the photon absorption
>> rate. It is easy to calculate if you know the extinction coefficient
>> of the dye and the irradiance and wavelength of light you're using.
>> See equation 2.1 of my paper here:
>>
>> Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E.
>> (2010).
>> Molecules and methods for super-resolution imaging. Methods in
>> Enzymology, 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3
>>
>> http://everydayscientist.com/pdfs/sjl16.pdf
>>
>> Alternatively, maybe a more practical metric would be to change the
>> illumination so that each sample has the same brightness on the
>> camera or the same signal to noise ratio or some other useful
>> quantity. Then record the bleaching half-time. That would tell you
>> exactly what you really want to know: given a certain starting image
>> quality, how long can I observe my sample? This measure should indeed
>> scale with the photobleaching quantum yield, but it also takes into
>> account the "brightness"
>> of the dye (i.e. the fluorescence quantum yield times the extinction
>> coefficient), as well as microscope factors like the chromatic
>> aberrations and camera quantum efficiency at different wavelengths.
>>
>> This is a pragmatic measure, but it would be a little hard to
>> directly compare completely different dyes, samples, microscopes,
>> etc. That's why I like absolute metrics, like the photobleaching
>> quantum yield and dye brightness.
>>
>> For a great reference on dye brightness, see:
>>
>> A guide to choosing fluorescent proteins
>> http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Me
>> thod s%20-%20Choosing%20fluorescent%20proteins.pdf
>>
>> (Note the measure that Shaner used for photo stability does indeed
>> take into account absorption rate: "Time for bleaching from an
>> initial emission rate of 1,000 photons/s down to 500 photons/s (t1/2;
>> for comparison, fluorescein at pH
>> 8.4 has
>> t1/2 of 5.2 s).")
>>
>> The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid
>> the pitfalls of multi-exponential fitting and reporting weighted
>> decay constants (see Lakowicz or equation 2.2 of my paper above).
>> Half-life is a super simple and pragmatic measure of sample
>> photobleaching.
>>
>>  On that note, can anybody recommend a good reference
>>> that discusses the subtleties of photobleaching?
>>>
>> As far as I know, there is no single review that really encompasses
>> all the subtleties of photobleaching. A lot of those subtleties are
>> not well understood.
>> People often blame singlet oxygen, but it's way more complicated than
>> that (e.g.
>> triplet/ground-state oxygen is an excellent triplet quencher, so it
>> is actually good to have around to reduce dyes in the triplet
>> bottleneck; see Hubner. J.
>> Chem. Phys.
>> 2001, 115, 9619). There are many paths to photobleaching, they are
>> difficult to observe, they yield a huge range of photoproducts, and
>> each dye is likely to have a completely different way it prefers to
>> die.
>>
>> There are some great examples of papers where the authors discovered
>> the photobleaching path of a particular dye or subset of dyes:
>>
>> Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
>> Photoswitching mechanism of cyanine dyes. Journal of the American
>> Chemical Society, 131, 18192-3.
>>
>> Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking
>> and switching behaviour of single molecules of green fluorescent protein.
>> Nature, 388,
>> 355-8.
>>
>> Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with
>> improved photostability. Organic Letters, 9, 2775-7.
>>
>> Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways
>> in
>> single-
>> molecule FRET experiments. Journal of the American Chemical Society,
>> 129,
>> 4643-
>> 54.
>>
>> Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of
>> Trolox as antiblinking and antibleaching reagent. Journal of the
>> American Chemical Society, 131, 5018-9.
>>
>> Berglund AJ. (2004). Nonexponential statistics of fluorescence
>> photobleaching. The Journal of Chemical Physics, 121, 2899-903.
>>
>> Best,
>>
>> -Sam
>>
>

[Gautier Papon]

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

Hello Michael,

The idea, at least for Argolight, is to provide solution to measure the reliability of your systems in the conditions you will use, so that you know there is no bias added by the system. Then of course, if your protocol has inherent fluctuation in the biological part, we cannot correct for it.


Le 23 mars 2015 à 17:14, Cammer, Michael <[hidden email]> a écrit :

> Am I missing something?
>
> I don’t understand how you can have a standard independent of the actual experimental condition since each fluorophore behaves differently and the specific biological/chemical environment may make a difference too.
>
> Also, you need to measure the illumination at the sample independently from the intensity on the detection side.
>
> Regards,
> Michael
>
> =========================================================================
> Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
>                          Cell:  914-309-3270     Temporary location:  SK2-7
>          http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew York
> Sent: Tuesday, March 17, 2015 4:36 PM
> To: [hidden email]
> Subject: Re: Photobleaching Standard
>
> *****
> 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 reasonable standardized sample to try:
> http://www.gattaquant.com/products/gatta-brightness.html
> I haven't tried them myself, but I'd like to. If anyone has, please tell me about your experience.
>
> This wouldn't directly tell you photobleaching rates, but since you actually know how many dye molecules you're starting with, it should make bleaching statistics simple to gather, and simple to interpret. It won't get at interesting sample-dependent bleaching questions, but it should help if you want to compare microscopes quantitatively.
>
> On Mon, Mar 16, 2015 at 1:26 PM, Neil Anthony <[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.
>> *****
>>
>> Cool, thanks Sam.  That's exactly the kind of answer I was looking
>> for; it's great to get the very specific details from an expert.
>>
>> Thanks
>> Neil
>>
>>
>>
>>
>> On 3/12/2015 2:40 PM, Sam Lord 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 Neil,
>>>
>>> Great question.
>>>
>>> On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[hidden email]>
>>> wrote:
>>>
>>>> I would presumably
>>>> calculate the intensity decay over a time lapse as a metric, but
>>>> wasn't sure if the sample type was a factor as I don't know much
>>>> about the photochemistry.
>>>>
>>> I have a pet peeve: when a paper reports photobleaching half-lives or
>>> bleaching decay times *without accounting for the rate of absorbing
>>> photons*.
>>> Photobleaching is a statistical process that typically occurs from
>>> the excited state of the molecule (e.g. from intersystem crossing to
>>> a triplet state and then further excitation and photodegratation) or
>>> from an excited producing singlet oxygen that then reacts with that
>>> molecule or a nearby molecule.
>>>
>>> Either way, the rate of photobleaching is going to depend on how
>>> frequently the dye cycles into the excited state. If you compare two
>>> samples without taking into account the excitation rate, you might
>>> think one sample is more susceptible to bleaching when really it is
>>> just brighter.
>>>
>>> I recommend using the photobleaching quantum yield as an absolute
>>> measure. It is the probability of photobleaching with each photon
>>> absorbed, or the ratio of the bleaching rate to the photon absorption
>>> rate. It is easy to calculate if you know the extinction coefficient
>>> of the dye and the irradiance and wavelength of light you're using.
>>> See equation 2.1 of my paper here:
>>>
>>> Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E.
>>> (2010).
>>> Molecules and methods for super-resolution imaging. Methods in
>>> Enzymology, 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3
>>>
>>> http://everydayscientist.com/pdfs/sjl16.pdf
>>>
>>> Alternatively, maybe a more practical metric would be to change the
>>> illumination so that each sample has the same brightness on the
>>> camera or the same signal to noise ratio or some other useful
>>> quantity. Then record the bleaching half-time. That would tell you
>>> exactly what you really want to know: given a certain starting image
>>> quality, how long can I observe my sample? This measure should indeed
>>> scale with the photobleaching quantum yield, but it also takes into
>>> account the "brightness"
>>> of the dye (i.e. the fluorescence quantum yield times the extinction
>>> coefficient), as well as microscope factors like the chromatic
>>> aberrations and camera quantum efficiency at different wavelengths.
>>>
>>> This is a pragmatic measure, but it would be a little hard to
>>> directly compare completely different dyes, samples, microscopes,
>>> etc. That's why I like absolute metrics, like the photobleaching
>>> quantum yield and dye brightness.
>>>
>>> For a great reference on dye brightness, see:
>>>
>>> A guide to choosing fluorescent proteins
>>> http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Me
>>> thod s%20-%20Choosing%20fluorescent%20proteins.pdf
>>>
>>> (Note the measure that Shaner used for photo stability does indeed
>>> take into account absorption rate: "Time for bleaching from an
>>> initial emission rate of 1,000 photons/s down to 500 photons/s (t1/2;
>>> for comparison, fluorescein at pH
>>> 8.4 has
>>> t1/2 of 5.2 s).")
>>>
>>> The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid
>>> the pitfalls of multi-exponential fitting and reporting weighted
>>> decay constants (see Lakowicz or equation 2.2 of my paper above).
>>> Half-life is a super simple and pragmatic measure of sample
>>> photobleaching.
>>>
>>> On that note, can anybody recommend a good reference
>>>> that discusses the subtleties of photobleaching?
>>>>
>>> As far as I know, there is no single review that really encompasses
>>> all the subtleties of photobleaching. A lot of those subtleties are
>>> not well understood.
>>> People often blame singlet oxygen, but it's way more complicated than
>>> that (e.g.
>>> triplet/ground-state oxygen is an excellent triplet quencher, so it
>>> is actually good to have around to reduce dyes in the triplet
>>> bottleneck; see Hubner. J.
>>> Chem. Phys.
>>> 2001, 115, 9619). There are many paths to photobleaching, they are
>>> difficult to observe, they yield a huge range of photoproducts, and
>>> each dye is likely to have a completely different way it prefers to
>>> die.
>>>
>>> There are some great examples of papers where the authors discovered
>>> the photobleaching path of a particular dye or subset of dyes:
>>>
>>> Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009).
>>> Photoswitching mechanism of cyanine dyes. Journal of the American
>>> Chemical Society, 131, 18192-3.
>>>
>>> Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking
>>> and switching behaviour of single molecules of green fluorescent protein.
>>> Nature, 388,
>>> 355-8.
>>>
>>> Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with
>>> improved photostability. Organic Letters, 9, 2775-7.
>>>
>>> Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways
>>> in
>>> single-
>>> molecule FRET experiments. Journal of the American Chemical Society,
>>> 129,
>>> 4643-
>>> 54.
>>>
>>> Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of
>>> Trolox as antiblinking and antibleaching reagent. Journal of the
>>> American Chemical Society, 131, 5018-9.
>>>
>>> Berglund AJ. (2004). Nonexponential statistics of fluorescence
>>> photobleaching. The Journal of Chemical Physics, 121, 2899-903.
>>>
>>> Best,
>>>
>>> -Sam
>>>
>>