Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647

[Neil Anthony]

*****
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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?


My example here is the difference between Alexa 633 and 647 using a 642 laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference in molecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is that the 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.


I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nm laser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?


Thanks for thinking on this splitting-of-hairs that really should just be done empirically :)


Neil



Collated details:


A647

EC - 270 kcm^-1M^-1 (WikiPedia)

EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)

Brightness @ 1x10^-1 MW cm^-2

- 3.5 x 10^4 Hz mol^-1

(Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)

QY - 0.33

lifetime - 1 ns


A633

EC - 159 kcm-1M-1 (WikiPedia)

EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)

Brightness @ 1x10^-1 MW cm^-2

- 1 x 10^4 Hz mol^-1

(Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)

QY ?

lifetime - 3.2 ns (https://www.thermofisher.com)







Neil Anthony, PhD  |  Assistant Scientist
Health Sciences Research Building (HSRB), Room EG21
1760 Haygood Drive, Atlanta, GA 30322

[hidden email]<mailto:[hidden email]>
ici.emory.edu<http://www.cores.emory.edu/ici/>
404 969-CORE


[http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>


________________________________

This e-mail message (including any attachments) is for the sole use of
the intended recipient(s) and may contain confidential and privileged
information. If the reader of this message is not the intended
recipient, you are hereby notified that any dissemination, distribution
or copying of this message (including any attachments) is strictly
prohibited.

If you have received this message in error, please contact
the sender by reply e-mail message and destroy all copies of the
original message (including attachments).

[Craig Brideau]

*****
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.
*****

We use Alexa 647 with a 638 and 640nm laser line (two different confocal
microscopes) and it has performed well. Empirically I have never noted any
exceptional amount of bleaching with this fluorophore, and generally found
it to be fairly robust overall.

Craig

On Thu, Jun 15, 2017 at 9:09 AM, Anthony, Neil <[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 was wondering if anybody knows the lesser of two evils: low extinction
> coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a 642
> laser line.  If you view these on a spectral viewer it's quite misleading.
> The normalized excitation spectra suggest that a 642 nm line would hit both
> the 633 and 647 with around 80% efficiency.  If you consider the brightness
> extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms
> of it's cross section (for those not familiar, think of it as a target,
> larger cross sections mean higher probabilities that the light will
> interact with the fluorophore), meaning you'll get 2-3 times more molecules
> excited with the same laser intensity/density.  The next step is how much
> of that light it will emit as fluorescence, measured as it's quantum yield
> (QY).  I can't find for 633, but considering the difference in molecular
> brightnesses measured with FCS I'd guess it's about the same if not a
> little more, guessing 0.3 - 0.4 range.  The last subtlety is that the 633
> dye will spend around 3x more in the excited state before fluorescing,
> lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what
> photophsics would help me make the decision between A633 and A647 on a
> 642nm laser line?  I know the A647 is less photostable, but if I have to
> use 3x less laser power for the same signal would I break even?
>
>
> Thanks for thinking on this splitting-of-hairs that really should just be
> done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist
> Health Sciences Research Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <
> http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/
> ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [
> http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <
> http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [
> http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <
> https://www.researchgate.net/profile/Neil_Anthony/>
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).
>

[Michael Giacomelli]

*****
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,

>I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?

If you have to use 3x the power but have 1/3 the absorption rate, then
you have no difference because only photons that are absorbed
contribute to bleaching.  Well assuming you aren't thermally damaging
the sample, which is unlikely at 633 nm.

Intuitively for two dyes with similar spectra and roughly comparable
absorption cross sections, I would expect the dye with the much longer
lifetime to work better, since a longer lifetime at a usable quantum
yield suggests a lower rate of nonradiative relaxations.  This is just
a rough rule of thumb though, no idea for your specific dyes.  Try
them and see?

Mike

On Thu, Jun 15, 2017 at 5:22 PM, Craig Brideau <[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.
> *****
>
> We use Alexa 647 with a 638 and 640nm laser line (two different confocal
> microscopes) and it has performed well. Empirically I have never noted any
> exceptional amount of bleaching with this fluorophore, and generally found
> it to be fairly robust overall.
>
> Craig
>
> On Thu, Jun 15, 2017 at 9:09 AM, Anthony, Neil <[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 was wondering if anybody knows the lesser of two evils: low extinction
>> coefficient (EC), or worse photostability?
>>
>>
>> My example here is the difference between Alexa 633 and 647 using a 642
>> laser line.  If you view these on a spectral viewer it's quite misleading.
>> The normalized excitation spectra suggest that a 642 nm line would hit both
>> the 633 and 647 with around 80% efficiency.  If you consider the brightness
>> extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms
>> of it's cross section (for those not familiar, think of it as a target,
>> larger cross sections mean higher probabilities that the light will
>> interact with the fluorophore), meaning you'll get 2-3 times more molecules
>> excited with the same laser intensity/density.  The next step is how much
>> of that light it will emit as fluorescence, measured as it's quantum yield
>> (QY).  I can't find for 633, but considering the difference in molecular
>> brightnesses measured with FCS I'd guess it's about the same if not a
>> little more, guessing 0.3 - 0.4 range.  The last subtlety is that the 633
>> dye will spend around 3x more in the excited state before fluorescing,
>> lifetime of 3.2 vs 1 ns.
>>
>>
>> I'm trying to wrap my head around the lesser of two evils, and what
>> photophsics would help me make the decision between A633 and A647 on a
>> 642nm laser line?  I know the A647 is less photostable, but if I have to
>> use 3x less laser power for the same signal would I break even?
>>
>>
>> Thanks for thinking on this splitting-of-hairs that really should just be
>> done empirically :)
>>
>>
>> Neil
>>
>>
>>
>> Collated details:
>>
>>
>> A647
>>
>> EC - 270 kcm^-1M^-1 (WikiPedia)
>>
>> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003,
>> http://www.jhc.org)
>>
>> Brightness @ 1x10^-1 MW cm^-2
>>
>> - 3.5 x 10^4 Hz mol^-1
>>
>> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
>> Tethered, Diaa Atta p68)
>>
>> QY - 0.33
>>
>> lifetime - 1 ns
>>
>>
>> A633
>>
>> EC - 159 kcm-1M-1 (WikiPedia)
>>
>> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003,
>> http://www.jhc.org)
>>
>> Brightness @ 1x10^-1 MW cm^-2
>>
>> - 1 x 10^4 Hz mol^-1
>>
>> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
>> Tethered, Diaa Atta p68)
>>
>> QY ?
>>
>> lifetime - 3.2 ns (https://www.thermofisher.com)
>>
>>
>>
>>
>>
>>
>>
>> Neil Anthony, PhD  |  Assistant Scientist
>> Health Sciences Research Building (HSRB), Room EG21
>> 1760 Haygood Drive, Atlanta, GA 30322
>>
>> [hidden email]<mailto:[hidden email]>
>> ici.emory.edu<http://www.cores.emory.edu/ici/>
>> 404 969-CORE
>>
>>
>> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <
>> http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/
>> ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [
>> http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <
>> http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [
>> http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <
>> https://www.researchgate.net/profile/Neil_Anthony/>
>>
>>
>> ________________________________
>>
>> This e-mail message (including any attachments) is for the sole use of
>> the intended recipient(s) and may contain confidential and privileged
>> information. If the reader of this message is not the intended
>> recipient, you are hereby notified that any dissemination, distribution
>> or copying of this message (including any attachments) is strictly
>> prohibited.
>>
>> If you have received this message in error, please contact
>> the sender by reply e-mail message and destroy all copies of the
>> original message (including attachments).
>>

[George McNamara]

*****
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,

the answer is: Yes. Oops, that may be for the greater of two unfavorable
properties (that is, equal). So to be clear: both are to be avoided
(unless CALI / FALI or DAB photooxiding).

What you probably really need is the quantum yields (QYs) of:

* fluorescence

* bad things

* heat transfer to surrounding molecules (which equals 1 = QY_fl - QYb.t.).

As for your QY_fl guess for AF633: not accounting for dark state(s) or
"dud" molecules (non fluorescent for other reasons, like the bottle is
not 100% AF633).

I encourage you to test your math and logic (and maybe some empiric FCS
and other F-techniques)  against fluorescein and tetrabromofluorescein
(aka eosin, see Tom Derrinck's use of eosin to photoconvert DAB into
nice precipitate).

enjoy,
George
wrt F-techniques, see

The F-techniques: advances in receptor protein studies.
Liu P, Ahmed S, Wohland T.
Trends Endocrinol Metab. 2008 Jul;19(5):181-90. doi:
10.1016/j.tem.2008.02.004. Epub 2008 Apr 1. Review.
PMID: 18387308


On 6/15/2017 11:09 AM, Anthony, Neil 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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a 642 laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference in molecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is that the 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nm laser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?
>
>
> Thanks for thinking on this splitting-of-hairs that really should just be done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist
> Health Sciences Research Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu (as of May 22, 2017)

[Claire Brown]

*****
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.
*****

Sometime ago a Molecular Probes rep told me the AF633 was not very soluble so they had a very hard time measuring the QY.
That is why they don't quote it on their webpage or in their literature.
She suspected it was much lower than AF647. We always recommend AF647.

I had remembered AF647 was more photostable but I see ThermoFisher doesn't have it on the dashboard. Not sure why.
https://www.thermofisher.com/ca/en/home/life-science/cell-analysis/fluorophores/alexa-fluor-647.html

Sincerely,

Claire

[Steffen Dietzel]

*****
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.
*****


And then, bleaching is dependent on the mounting medium you use. Just to
add to the parameters to look at. What works in one anti-fade medium
does not necessarily work in another.

I guess this is one of the very few cases where it actually might be
quicker to do the experiment rather than to go to the library.

Steffen

Am 15.06.2017 um 17:09 schrieb Anthony, Neil:

> *****
> 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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a 642laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference inmolecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is thatthe 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nmlaser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?
>
>
> Thanks for thinking on this splitting-of-hairs that really should just be done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist
> Health Sciences Research Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).
>

--
-- ----------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat.
Head of the Core Facility Bioimaging at the Biomedical Center
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für Experimentelle Medizin

Address:
Biomedical Center
Großhaderner Straße 9
D-82152 Planegg-Martinsried

Phone: [phone]
skype: steffendietzel
e-mail: [hidden email]
fax-to-e-mail: +49/89/2180-9971518
http://www.bioimaging.bmc.med.uni-muenchen.de



--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Neil Anthony]

*****
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.
*****


Thanks all for your thoughts on A633 vs A647 :)


Some great tips, and many avenues I hadn't thought of.


How I love the Confocal Listserv!


I bid you good science.


Neil



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Steffen Dietzel <[hidden email]>
Sent: Monday, June 19, 2017 5:55:13 AM
To: [hidden email]
Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647

*****
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.
*****


And then, bleaching is dependent on the mounting medium you use. Just to
add to the parameters to look at. What works in one anti-fade medium
does not necessarily work in another.

I guess this is one of the very few cases where it actually might be
quicker to do the experiment rather than to go to the library.

Steffen

Am 15.06.2017 um 17:09 schrieb Anthony, Neil:

> *****
> 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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a 642laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference inmolecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is thatthe 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nmlaser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?
>
>
> Thanks for thinking on this splitting-of-hairs that really should just be done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699–1712, 2003, http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist
> Health Sciences Research Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination, distribution
> or copying of this message (including any attachments) is strictly
> prohibited.
>
> If you have received this message in error, please contact
> the sender by reply e-mail message and destroy all copies of the
> original message (including attachments).
>

--
-- ----------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat.
Head of the Core Facility Bioimaging at the Biomedical Center
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für Experimentelle Medizin

Address:
Biomedical Center
Großhaderner Straße 9
D-82152 Planegg-Martinsried

Phone: [phone]
skype: steffendietzel
e-mail: [hidden email]
fax-to-e-mail: +49/89/2180-9971518
http://www.bioimaging.bmc.med.uni-muenchen.de



--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Paul Rigby-2]

*****
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 agree, the Confocal Listserver is an amazing resource...

One small question that I have in relation to this discussion about AF633 and AF647:

In the Mol Probes/Thermo/Life Tech tutorials on fluorescence, it is stated the photodamage/photobleaching primary occurs when a additional photon hits a fluorophore when it is in its semi-stable excited state.

Therefore:
1. If you use lower laser power, there is a lower probability of this occurring (therefore less photobleaching);
2. If you use a shorter pixel dwell time, there is also a lower probability of this happening. Is this why spinning disks cause less photobleaching?

Would it therefore follow (and discounting many of the other variables), if a fluorophore had a shorter lifetime (it spends less time in the semi-stable excited state), that fluorophore will be more photostable?
If this logic follows, then AF647 (with a lifetime of 1ns) should be more photostable that AF633 (with a lifetime of 3.2ns).

This seems too simple and have I missed something significant here? All views appreciated.
Cheers
Paul

Assoc. Prof. Paul Rigby
Optical Microscopy Specialist
Centre for Microscopy, Characterisation & Analysis (M519)
The University of Western Australia
35 Stirling Highway
Perth  WA  6009
Australia


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Anthony, Neil
Sent: Monday, 19 June 2017 9:52 PM
To: [hidden email]
Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647

*****
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.
*****


Thanks all for your thoughts on A633 vs A647 :)


Some great tips, and many avenues I hadn't thought of.


How I love the Confocal Listserv!


I bid you good science.


Neil



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Steffen Dietzel <[hidden email]>
Sent: Monday, June 19, 2017 5:55:13 AM
To: [hidden email]
Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647

*****
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.
*****


And then, bleaching is dependent on the mounting medium you use. Just to add to the parameters to look at. What works in one anti-fade medium does not necessarily work in another.

I guess this is one of the very few cases where it actually might be quicker to do the experiment rather than to go to the library.

Steffen

Am 15.06.2017 um 17:09 schrieb Anthony, Neil:

> *****
> 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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a 642laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference inmolecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is thatthe 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nmlaser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?
>
>
> Thanks for thinking on this splitting-of-hairs that really should just
> be done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist Health Sciences Research
> Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>
>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination,
> distribution or copying of this message (including any attachments) is
> strictly prohibited.
>
> If you have received this message in error, please contact the sender
> by reply e-mail message and destroy all copies of the original message
> (including attachments).
>

--
-- ----------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat.
Head of the Core Facility Bioimaging at the Biomedical Center Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für Experimentelle Medizin

Address:
Biomedical Center
Großhaderner Straße 9
D-82152 Planegg-Martinsried

Phone: [phone]
skype: steffendietzel
e-mail: [hidden email]
fax-to-e-mail: +49/89/2180-9971518
http://www.bioimaging.bmc.med.uni-muenchen.de



--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Aryeh Weiss]

*****
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.
*****

On 21/06/2017 8:16, Paul Rigby 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 agree, the Confocal Listserver is an amazing resource...
>
> One small question that I have in relation to this discussion about AF633 and AF647:
>
> In the Mol Probes/Thermo/Life Tech tutorials on fluorescence, it is stated the photodamage/photobleaching primary occurs when a additional photon hits a fluorophore when it is in its semi-stable excited state.

Does this imply that photobleaching should be a function of intensity
squared? Is this in fact the case?

> Therefore:
> 1. If you use lower laser power, there is a lower probability of this occurring (therefore less photobleaching);
> 2. If you use a shorter pixel dwell time, there is also a lower probability of this happening. Is this why spinning disks cause less photobleaching?
>
> Would it therefore follow (and discounting many of the other variables), if a fluorophore had a shorter lifetime (it spends less time in the semi-stable excited state), that fluorophore will be more photostable?
> If this logic follows, then AF647 (with a lifetime of 1ns) should be more photostable that AF633 (with a lifetime of 3.2ns).
>
> This seems too simple and have I missed something significant here? All views appreciated.
> Cheers
> Paul
>
> Assoc. Prof. Paul Rigby
> Optical Microscopy Specialist
> Centre for Microscopy, Characterisation & Analysis (M519)
> The University of Western Australia
> 35 Stirling Highway
> Perth  WA  6009
> Australia
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Anthony, Neil
> Sent: Monday, 19 June 2017 9:52 PM
> To: [hidden email]
> Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647
>
> *****
> 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.
> *****
>
>
> Thanks all for your thoughts on A633 vs A647 :)
>
>
> Some great tips, and many avenues I hadn't thought of.
>
>
> How I love the Confocal Listserv!
>
>
> I bid you good science.
>
>
> Neil
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Steffen Dietzel <[hidden email]>
> Sent: Monday, June 19, 2017 5:55:13 AM
> To: [hidden email]
> Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa 633 vs 647
>
> *****
> 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.
> *****
>
>
> And then, bleaching is dependent on the mounting medium you use. Just to add to the parameters to look at. What works in one anti-fade medium does not necessarily work in another.
>
> I guess this is one of the very few cases where it actually might be quicker to do the experiment rather than to go to the library.
>
> Steffen
>
> Am 15.06.2017 um 17:09 schrieb Anthony, Neil:
>> *****
>> 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 was wondering if anybody knows the lesser of two evils: low extinction coefficient (EC), or worse photostability?
>>
>>
>> My example here is the difference between Alexa 633 and 647 using a 642laser line.  If you view these on a spectral viewer it's quite misleading.  The normalized excitation spectra suggest that a 642 nm line would hit both the 633 and 647 with around 80% efficiency.  If you consider the brightness extinction coefficient you'll see that 647 is 2-3 times 'larger' in terms of it's cross section (for those not familiar, think of it as a target, larger cross sections mean higher probabilities that the light will interact with the fluorophore), meaning you'll get 2-3 times more molecules excited with the same laser intensity/density.  The next step is how much of that light it will emit as fluorescence, measured as it's quantum yield (QY).  I can't find for 633, but considering the difference inmolecular brightnesses measured with FCS I'd guess it's about the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety is thatthe 633 dye will spend around 3x more in the excited state before fluorescing, lifetime of 3.2 vs 1 ns.
>>
>>
>> I'm trying to wrap my head around the lesser of two evils, and what photophsics would help me make the decision between A633 and A647 on a 642nmlaser line?  I know the A647 is less photostable, but if I have to use 3x less laser power for the same signal would I break even?
>>
>>
>> Thanks for thinking on this splitting-of-hairs that really should just
>> be done empirically :)
>>
>>
>> Neil
>>
>>
>>
>> Collated details:
>>
>>
>> A647
>>
>> EC - 270 kcm^-1M^-1 (WikiPedia)
>>
>> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
>> http://www.jhc.org)
>>
>> Brightness @ 1x10^-1 MW cm^-2
>>
>> - 3.5 x 10^4 Hz mol^-1
>>
>> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
>> Tethered, Diaa Atta p68)
>>
>> QY - 0.33
>>
>> lifetime - 1 ns
>>
>>
>> A633
>>
>> EC - 159 kcm-1M-1 (WikiPedia)
>>
>> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
>> http://www.jhc.org)
>>
>> Brightness @ 1x10^-1 MW cm^-2
>>
>> - 1 x 10^4 Hz mol^-1
>>
>> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
>> Tethered, Diaa Atta p68)
>>
>> QY ?
>>
>> lifetime - 3.2 ns (https://www.thermofisher.com)
>>
>>
>>
>>
>>
>>
>>
>> Neil Anthony, PhD  |  Assistant Scientist Health Sciences Research
>> Building (HSRB), Room EG21
>> 1760 Haygood Drive, Atlanta, GA 30322
>>
>> [hidden email]<mailto:[hidden email]>
>> ici.emory.edu<http://www.cores.emory.edu/ici/>
>> 404 969-CORE
>>
>>
>> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <https://www.researchgate.net/profile/Neil_Anthony/>
>>
>>
>> ________________________________
>>
>> This e-mail message (including any attachments) is for the sole use of
>> the intended recipient(s) and may contain confidential and privileged
>> information. If the reader of this message is not the intended
>> recipient, you are hereby notified that any dissemination,
>> distribution or copying of this message (including any attachments) is
>> strictly prohibited.
>>
>> If you have received this message in error, please contact the sender
>> by reply e-mail message and destroy all copies of the original message
>> (including attachments).
>>
> --
> -- ----------------------------------------------------------
>
> Steffen Dietzel, PD Dr. rer. nat.
> Head of the Core Facility Bioimaging at the Biomedical Center Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für Experimentelle Medizin
>
> Address:
> Biomedical Center
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
>
> Phone: [phone]
> skype: steffendietzel
> e-mail: [hidden email]
> fax-to-e-mail: +49/89/2180-9971518
> http://www.bioimaging.bmc.med.uni-muenchen.de
>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Johan Herz]

*****
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.
*****

Paul,

Should you not also take into account the quantum yield of the fluorophore?
What are the yields of AF647 and AF633?

Best regards,

Johan Herz, Sales Engineer

Please visit FLIM.camera for more information about the Toggel.

Tel: [phone] | Skype: lambert-johan
Lambert Instruments BV|Leonard Springerlaan 19 (5th floor)|9727 KB
Groningen|The Netherlands
Dutch Chamber of Commerce nr.: 52396940 | www.lambertinstruments.com

2017-06-21 7:16 GMT+02:00 Paul Rigby <[hidden email]>:

> *****
> 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 agree, the Confocal Listserver is an amazing resource...
>
> One small question that I have in relation to this discussion about AF633
> and AF647:
>
> In the Mol Probes/Thermo/Life Tech tutorials on fluorescence, it is stated
> the photodamage/photobleaching primary occurs when a additional photon hits
> a fluorophore when it is in its semi-stable excited state.
>
> Therefore:
> 1. If you use lower laser power, there is a lower probability of this
> occurring (therefore less photobleaching);
> 2. If you use a shorter pixel dwell time, there is also a lower
> probability of this happening. Is this why spinning disks cause less
> photobleaching?
>
> Would it therefore follow (and discounting many of the other variables),
> if a fluorophore had a shorter lifetime (it spends less time in the
> semi-stable excited state), that fluorophore will be more photostable?
> If this logic follows, then AF647 (with a lifetime of 1ns) should be more
> photostable that AF633 (with a lifetime of 3.2ns).
>
> This seems too simple and have I missed something significant here? All
> views appreciated.
> Cheers
> Paul
>
> Assoc. Prof. Paul Rigby
> Optical Microscopy Specialist
> Centre for Microscopy, Characterisation & Analysis (M519)
> The University of Western Australia
> 35 Stirling Highway
> Perth  WA  6009
> Australia
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Anthony, Neil
> Sent: Monday, 19 June 2017 9:52 PM
> To: [hidden email]
> Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa
> 633 vs 647
>
> *****
> 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.
> *****
>
>
> Thanks all for your thoughts on A633 vs A647 :)
>
>
> Some great tips, and many avenues I hadn't thought of.
>
>
> How I love the Confocal Listserv!
>
>
> I bid you good science.
>
>
> Neil
>
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on
> behalf of Steffen Dietzel <[hidden email]>
> Sent: Monday, June 19, 2017 5:55:13 AM
> To: [hidden email]
> Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa
> 633 vs 647
>
> *****
> 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.
> *****
>
>
> And then, bleaching is dependent on the mounting medium you use. Just to
> add to the parameters to look at. What works in one anti-fade medium does
> not necessarily work in another.
>
> I guess this is one of the very few cases where it actually might be
> quicker to do the experiment rather than to go to the library.
>
> Steffen
>
> Am 15.06.2017 um 17:09 schrieb Anthony, Neil:
> > *****
> > 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 was wondering if anybody knows the lesser of two evils: low extinction
> coefficient (EC), or worse photostability?
> >
> >
> > My example here is the difference between Alexa 633 and 647 using a
> 642laser line.  If you view these on a spectral viewer it's quite
> misleading.  The normalized excitation spectra suggest that a 642 nm line
> would hit both the 633 and 647 with around 80% efficiency.  If you consider
> the brightness extinction coefficient you'll see that 647 is 2-3 times
> 'larger' in terms of it's cross section (for those not familiar, think of
> it as a target, larger cross sections mean higher probabilities that the
> light will interact with the fluorophore), meaning you'll get 2-3 times
> more molecules excited with the same laser intensity/density.  The next
> step is how much of that light it will emit as fluorescence, measured as
> it's quantum yield (QY).  I can't find for 633, but considering the
> difference inmolecular brightnesses measured with FCS I'd guess it's about
> the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety
> is thatthe 633 dye will spend around 3x more in the excited state before
> fluorescing, lifetime of 3.2 vs 1 ns.
> >
> >
> > I'm trying to wrap my head around the lesser of two evils, and what
> photophsics would help me make the decision between A633 and A647 on a
> 642nmlaser line?  I know the A647 is less photostable, but if I have to use
> 3x less laser power for the same signal would I break even?
> >
> >
> > Thanks for thinking on this splitting-of-hairs that really should just
> > be done empirically :)
> >
> >
> > Neil
> >
> >
> >
> > Collated details:
> >
> >
> > A647
> >
> > EC - 270 kcm^-1M^-1 (WikiPedia)
> >
> > EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> > http://www.jhc.org)
> >
> > Brightness @ 1x10^-1 MW cm^-2
> >
> > - 3.5 x 10^4 Hz mol^-1
> >
> > (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> > Tethered, Diaa Atta p68)
> >
> > QY - 0.33
> >
> > lifetime - 1 ns
> >
> >
> > A633
> >
> > EC - 159 kcm-1M-1 (WikiPedia)
> >
> > EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> > http://www.jhc.org)
> >
> > Brightness @ 1x10^-1 MW cm^-2
> >
> > - 1 x 10^4 Hz mol^-1
> >
> > (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> > Tethered, Diaa Atta p68)
> >
> > QY ?
> >
> > lifetime - 3.2 ns (https://www.thermofisher.com)
> >
> >
> >
> >
> >
> >
> >
> > Neil Anthony, PhD  |  Assistant Scientist Health Sciences Research
> > Building (HSRB), Room EG21
> > 1760 Haygood Drive, Atlanta, GA 30322
> >
> > [hidden email]<mailto:[hidden email]>
> > ici.emory.edu<http://www.cores.emory.edu/ici/>
> > 404 969-CORE
> >
> >
> > [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <
> http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/
> ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [
> http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <
> http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [
> http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <
> https://www.researchgate.net/profile/Neil_Anthony/>
> >
> >
> > ________________________________
> >
> > This e-mail message (including any attachments) is for the sole use of
> > the intended recipient(s) and may contain confidential and privileged
> > information. If the reader of this message is not the intended
> > recipient, you are hereby notified that any dissemination,
> > distribution or copying of this message (including any attachments) is
> > strictly prohibited.
> >
> > If you have received this message in error, please contact the sender
> > by reply e-mail message and destroy all copies of the original message
> > (including attachments).
> >
>
> --
> -- ----------------------------------------------------------
>
> Steffen Dietzel, PD Dr. rer. nat.
> Head of the Core Facility Bioimaging at the Biomedical Center
> Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
> Experimentelle Medizin
>
> Address:
> Biomedical Center
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
>
> Phone: [phone]
> skype: steffendietzel
> e-mail: [hidden email]
> fax-to-e-mail: +49/89/2180-9971518
> http://www.bioimaging.bmc.med.uni-muenchen.de
>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany
>
> http://www.bioimaging.bmc.med.uni-muenchen.de
>

[Craig Brideau]

*****
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.
*****

Fluorophores have a lifetime over which they will remain in the excited
state before emitting. In theory fluorophores with long lifetimes are more
vulnerable to photobleaching as the molecule stays in the excited state
longer. This increases the probability of interaction with an additional
excitation photon while in that state, which can open up energy pathways
that lead to the bleaching of the molecule rather than fluorescence
emission. In practice, other factors also influence this, such as oxygen in
the environment that can lead to free radical production (possible for UV
or 2P scenarios) which can destroy the fluorophore through reduction
interactions.
Another problem is that the absorption profile of the molecule for the
excited state may be different from the relaxed state, so the
wavelength/energy you need to get from the excited state to a possibly
destructive state is not necessarily what you need to get from the ground
to the excited state.
Hope this helped more than it confused. Molecular lifetime is a fairly
complex topic!

Craig

On Jun 20, 2017 11:57 PM, "Paul Rigby" <[hidden email]> wrote:


In the Mol Probes/Thermo/Life Tech tutorials on fluorescence, it is stated
the photodamage/photobleaching primary occurs when a additional photon hits
a fluorophore when it is in its semi-stable excited state.

Therefore:
1. If you use lower laser power, there is a lower probability of this
occurring (therefore less photobleaching);
2. If you use a shorter pixel dwell time, there is also a lower probability
of this happening. Is this why spinning disks cause less photobleaching?

Would it therefore follow (and discounting many of the other variables), if
a fluorophore had a shorter lifetime (it spends less time in the
semi-stable excited state), that fluorophore will be more photostable?
If this logic follows, then AF647 (with a lifetime of 1ns) should be more
photostable that AF633 (with a lifetime of 3.2ns).

This seems too simple and have I missed something significant here? All
views appreciated.
Cheers
Paul

Assoc. Prof. Paul Rigby
Optical Microscopy Specialist
Centre for Microscopy, Characterisation & Analysis (M519)
The University of Western Australia
35 Stirling Highway
Perth  WA  6009
Australia


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Anthony, Neil
Sent: Monday, 19 June 2017 9:52 PM
To: [hidden email]
Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa
633 vs 647

*****
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.
*****


Thanks all for your thoughts on A633 vs A647 :)


Some great tips, and many avenues I hadn't thought of.


How I love the Confocal Listserv!


I bid you good science.


Neil



________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of Steffen Dietzel <[hidden email]>
Sent: Monday, June 19, 2017 5:55:13 AM
To: [hidden email]
Subject: Re: Fluorescence Extinction Coefficient vs Photostability - Alexa
633 vs 647

*****
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.
*****


And then, bleaching is dependent on the mounting medium you use. Just to
add to the parameters to look at. What works in one anti-fade medium does
not necessarily work in another.

I guess this is one of the very few cases where it actually might be
quicker to do the experiment rather than to go to the library.

Steffen

Am 15.06.2017 um 17:09 schrieb Anthony, Neil:
> *****
> 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 was wondering if anybody knows the lesser of two evils: low extinction
coefficient (EC), or worse photostability?
>
>
> My example here is the difference between Alexa 633 and 647 using a
642laser line.  If you view these on a spectral viewer it's quite
misleading.  The normalized excitation spectra suggest that a 642 nm line
would hit both the 633 and 647 with around 80% efficiency.  If you consider
the brightness extinction coefficient you'll see that 647 is 2-3 times
'larger' in terms of it's cross section (for those not familiar, think of
it as a target, larger cross sections mean higher probabilities that the
light will interact with the fluorophore), meaning you'll get 2-3 times
more molecules excited with the same laser intensity/density.  The next
step is how much of that light it will emit as fluorescence, measured as
it's quantum yield (QY).  I can't find for 633, but considering the
difference inmolecular brightnesses measured with FCS I'd guess it's about
the same if not a little more, guessing 0.3 - 0.4 range.  The last subtlety
is thatthe 633 dye will spend around 3x more in the excited state before
fluorescing, lifetime of 3.2 vs 1 ns.
>
>
> I'm trying to wrap my head around the lesser of two evils, and what
photophsics would help me make the decision between A633 and A647 on a
642nmlaser line?  I know the A647 is less photostable, but if I have to use
3x less laser power for the same signal would I break even?

>
>
> Thanks for thinking on this splitting-of-hairs that really should just
> be done empirically :)
>
>
> Neil
>
>
>
> Collated details:
>
>
> A647
>
> EC - 270 kcm^-1M^-1 (WikiPedia)
>
> EC - 240 kcm^-1M^-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 3.5 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY - 0.33
>
> lifetime - 1 ns
>
>
> A633
>
> EC - 159 kcm-1M-1 (WikiPedia)
>
> EC - 100 kcm-1M-1 (Berlier etal, Volume 51(12): 1699-1712, 2003,
> http://www.jhc.org)
>
> Brightness @ 1x10^-1 MW cm^-2
>
> - 1 x 10^4 Hz mol^-1
>
> (Time Resolved Single Molecule Fluorescence Spectroscopy on Surface
> Tethered, Diaa Atta p68)
>
> QY ?
>
> lifetime - 3.2 ns (https://www.thermofisher.com)
>
>
>
>
>
>
>
> Neil Anthony, PhD  |  Assistant Scientist Health Sciences Research
> Building (HSRB), Room EG21
> 1760 Haygood Drive, Atlanta, GA 30322
>
> [hidden email]<mailto:[hidden email]>
> ici.emory.edu<http://www.cores.emory.edu/ici/>
> 404 969-CORE
>
>
> [http://www.cores.emory.edu/ici/images/EU_ICIC_EICF_SmlLogo.png] <

http://www.cores.emory.edu/ici/>      [http://www.cores.emory.edu/
ici/images/facebook_logo.png]  <https://www.facebook.com/ICIEmory>  [
http://www.cores.emory.edu/ici/images/linkedin_logo.png]  <
http://www.linkedin.com/pub/neil-anthony/35/a0b/126>        [
http://www.cores.emory.edu/ici/images/researchgate_logo.png]  <
https://www.researchgate.net/profile/Neil_Anthony/>

>
>
> ________________________________
>
> This e-mail message (including any attachments) is for the sole use of
> the intended recipient(s) and may contain confidential and privileged
> information. If the reader of this message is not the intended
> recipient, you are hereby notified that any dissemination,
> distribution or copying of this message (including any attachments) is
> strictly prohibited.
>
> If you have received this message in error, please contact the sender
> by reply e-mail message and destroy all copies of the original message
> (including attachments).
>

--
-- ----------------------------------------------------------

Steffen Dietzel, PD Dr. rer. nat.
Head of the Core Facility Bioimaging at the Biomedical Center
Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für
Experimentelle Medizin

Address:
Biomedical Center
Großhaderner Straße 9
D-82152 Planegg-Martinsried

Phone: [phone]
skype: steffendietzel
e-mail: [hidden email]
fax-to-e-mail: +49/89/2180-9971518
http://www.bioimaging.bmc.med.uni-muenchen.de



--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

*****
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.
*****

Aurox Ltd are actively looking for an R&D Software Engineer to join our innovative and friendly team at our headquarters in Oxfordshire.

The candidate will primarily be responsible for designing and building software to power the confocal instruments built by Aurox.  In practice this will mean work on both R&D and production systems. The role will also involve communicating directly with business partners and clients to understand their requirements and assisting in product demonstrations and trade shows.  You will be based at the Aurox headquarters in Culham Science Centre near Oxford, UK.

The company will provide competitive salary depending on qualifications and experience. As a dynamic spin-off company Aurox will present opportunities for quick professional progression for a suitably motivated person.

Principal duties:
- Supporting the Aurox Visionary software, providing feature updates and bugfixes.
- Writing new application software for next-generation devices.
- Implementing new features in device firmware, and writing new firmware as part of the research process.
- Writing tools for production

Essential skills:
- Experience in C/C++ and Qt
- Familiarity with Linux/Windows/OSX
- Experience with GPU processing (CUDA, OpenCL)
- Knowledge of embedded systems programming
- Experience with image processing

Desirable:
- PhD in scientific field
- Experience with optics or microscopy.
- Familiarity with PIC/PIC32 chipset
- Some knowledge of Java/Python/Javascript

If you are interested in this varied and interesting position, please send your CV and a covering letter to [hidden email]

I look forward to hearing from you

Phillipa


Phillipa Timmins

Head of Sales
Aurox Ltd

Culham Science Centre
Abingdon
OX14 3DB

Tel: [phone]
Mob: [phone]