Ratiometric measurements for multiphoton microscopy

[Konstantín Levitskiy]

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Dear all.

 

One of our groups is interested in doing fluorescence ratiometric
measurements (e.g., FURA-2, ROS) using a multiphoton microscopy system. From
what we understand the laser tuning speeds won’t be fast enough to switch
between two different excitation wavelengths, and we were wondering if there
could be ways to maintain these ratiometric attributes while still
performing a laser two-photon excitation. Any ideas or suggestions will be
very much appreciated.

 

Thanks and regard,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: [hidden email]

Web: www.ibis-sevilla.es

 

 

AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y
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para propósito alguno, ni divulgar su contenido a ninguna persona.

P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir
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[mmodel]

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Konstantin,
I would look probes that have common excitation and a shift in emission (Indo-1 for example)

Mike Model

________________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Microscopia-IBIS <[hidden email]>
Sent: Monday, April 25, 2016 5:32 AM
To: [hidden email]
Subject: Ratiometric measurements for multiphoton microscopy

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Dear all.



One of our groups is interested in doing fluorescence ratiometric
measurements (e.g., FURA-2, ROS) using a multiphoton microscopy system. From
what we understand the laser tuning speeds won’t be fast enough to switch
between two different excitation wavelengths, and we were wondering if there
could be ways to maintain these ratiometric attributes while still
performing a laser two-photon excitation. Any ideas or suggestions will be
very much appreciated.



Thanks and regard,

Dr. Konstantín Levitskiy

Servicio de Microscopía

InstitutodeBiomedicinadeSevilla - IBiS

Campus del Hospital Universitario Virgen del Rocío

Avda. Manuel Siurot s/nº

41013 Sevilla

Tlfno: 955 92 3030

Email: [hidden email]

Web: www.ibis-sevilla.es





AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y
pueden estar protegidos por disposiciones legales. Si Vd. no es el
destinatario del mismo, por favor, notifíquenoslo inmediatamente y destruya
o devuelva el original. No deberá copiar este mensaje ni sus anexos o usarlo
para propósito alguno, ni divulgar su contenido a ninguna persona.

P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir
este mensaje, compruebe que sea necesario hacerlo.

[Johannes Helm]

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Dear Konstantin,

we had done something like the thing you describe for a CLSM in the 90s.
Please, check these papers:

A)
P.J. Helm, O. Franksson, and K. Carlsson (1995),
A confocal scanning laser microscope for quantitative ratiometric 3D
measurements of [Ca2+] and Ca2+ diffusions in living cells stained
with Fura-2,
Pflügers Archiv - European Journal of Physiology, Vol. 429, pp. 672-681

B)
PJ Helm, A Patwardhan, and EMM Manders (1997),
A study of the precision of confocal, ratiometric, Fura-2 based [Ca2+]
measurements,
Cell Calcium 22(4):287-298


These systems might work even in the multi photon case.

HOWEVER:
Concerning A)
1)
Using excitation ratiometric dyes will be troublesome in the multi
photon case:
a)
It is difficult, often impossible, to avoid cross excitation in
non-linear excitation. An exception is the CFP-YFP-FRET based Chameleont
dye. However, you would need TWO lasers for this dye, which would be
rather expensive and space consuming ("stapling" lasers is certainy not
recommended, however, you could install them on a sub-table-top shelf,
which some Companies selling and producing Optical tables offer).
b)
Whe using the Chameleont dye, you would need a FRET detection. You might
check another paper with a proposal on how to measure FRET:

Helm P.J. (2012),
Proposal of a New Method for Measuring Foerster Resonance Energy
Transfer (FRET) Rapidly, Quantitatively and Non-Destructively,
International Journal of Molecular Sciences 13(10):12367-12382

c)
Using an emission ratiometic dye might be technically easier, although I
know that one needs "this dye" and cannot use "that one" for some
reasons.

2)
You would have to carefully select your EOMs (or AOMs)!

Concerning B)
WARNING:
One needs a really large number of detected photons in order to be able
to use the raw data as basis for a fully quantitative ratiometric
analysis.


Please, note that Kjell Carlsson and Anders Liljeborg had continued to
work on the  IMS method, which is a beautiful technology, indeed. It can
be used - and has been so, although not for ratiometric imaging, for
which it nevertheless would be suitable - for suppressing cross talk and
bleed through between different imaging channels. I also allow myself to
suggest this, in my opinion outstanding, paper,

Carlsson K1, Liljeborg A.
Simultaneous confocal lifetime imaging of multiple fluorophores using
the intensity-modulated multiple-wavelength scanning (IMS) technique.
J Microsc. 1998 Aug;191(2):119-127.

Best wishes,

Johannes

--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
University of Oslo
Institute of Basic Medical Sciences
Department of Molecular Medicine
Division of Physiology
P. O. Box 1103 - Blindern
NO-0317 Oslo
Norway

Voice: [phone]
Fax: [phone]



On 2016-04-25 11:32, Microscopia-IBIS wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your
> posting.
> *****
>
> Dear all.
>
>
>
> One of our groups is interested in doing fluorescence ratiometric
> measurements (e.g., FURA-2, ROS) using a multiphoton microscopy system.
> From
> what we understand the laser tuning speeds won't be fast enough to
> switch
> between two different excitation wavelengths, and we were wondering if
> there
> could be ways to maintain these ratiometric attributes while still
> performing a laser two-photon excitation. Any ideas or suggestions will
> be
> very much appreciated.
>
>
>
> Thanks and regard,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email: [hidden email]
>
> Web: www.ibis-sevilla.es
>
>
>
>
>
> AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son
> confidenciales y
> pueden estar protegidos por disposiciones legales. Si Vd. no es el
> destinatario del mismo, por favor, notifíquenoslo inmediatamente y
> destruya
> o devuelva el original. No deberá copiar este mensaje ni sus anexos o
> usarlo
> para propósito alguno, ni divulgar su contenido a ninguna persona.
>
> P El medio ambiente es nuestra responsabilidad. Por favor, antes de
> imprimir
> este mensaje, compruebe que sea necesario hacerlo.

WWW: folk.uio.no/jhelm

[Christian Wilms]

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Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.

Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.

Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.

FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.

An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.

If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)

Good luck with your experiments!

[Christian Wilms]

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Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.

Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.

Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.

FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.

An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.

If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)

Good luck with your experiments!

Best, Christian

[Mark Cannell-2]

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Hi All

When we used info-1 with 690 nm 2P excitation, I thought that the ratio image was quite reasonable (see Jones, K.T., Soeller, C., Cannell, M.B., 1998. The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627–4635). Also, using a second Ca insensitive dye to construct a “ratio" measurement is unwise -the point of the second wavelength is to control for bleaching and dye distribution etc.
 my 2c

Cheers


On 26/04/2016, at 10:27 am, Christian Wilms <[hidden email]> wrote:

>
> Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.
>
> Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.
>
> Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.
>
> FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.
>
> An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.
>
> If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)
>
> Good luck with your experiments!

Mark  B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Christian Wilms]

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"When we used info-1 with 690 nm 2P excitation, I thought that the ratio image was quite reasonable (see Jones, K.T., Soeller, C., Cannell, M.B., 1998. The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627-4635)."

I admit that I never tried it in cells, only cuvettes. But I could reproduce the more than an order of magnitude lower excitation efficiency than e.g. Fura-2 (http://www.drbio.cornell.edu/cross_sections.html) and decided that the required excitation power was not ideal for my experiments on small neuronal compartments. When imaging larger structures at low zoom, that should be much less of a problem.

"Also, using a second Ca insensitive dye to construct a "ratio" measurement is unwise -the point of the second wavelength is to control for bleaching and dye distribution etc."

I am also not at all a fan of the approach. It comes with numerous problems, starting with different diffusion rates of the two dyes over different bleaching behaviour, to differential scattering of the two emission wavelengths, leading to a depth-dependent gradient in the ratio, that would need to be corrected for. Having said all that, it has become pretty much standard in neuroscience to report 'green over red' ratios. See publications by the Sabatini for examples.

Best, Chris

[Mark Cannell-2]

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Hi Chris

Any idea what is the cause of the difference between the measurements you cite and the conclusions of Szmacinski et al (Photochem Photobiol. 1993 Sep;58(3):341-5): "The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4 s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. “?

Cheers Mark


.
Calcium-dependent fluorescence lifetimes of Indo-1 for one- and two-photon excitation of fluorescence.
Szmacinski H1, Gryczynski I, Lakowicz JR.
On 27/04/2016, at 11:25 am, Christian Wilms <[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.
> *****
>
> "When we used info-1 with 690 nm 2P excitation, I thought that the ratio image was quite reasonable (see Jones, K.T., Soeller, C., Cannell, M.B., 1998. The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627-4635)."
>
> I admit that I never tried it in cells, only cuvettes. But I could reproduce the more than an order of magnitude lower excitation efficiency than e.g. Fura-2 (http://www.drbio.cornell.edu/cross_sections.html) and decided that the required excitation power was not ideal for my experiments on small neuronal compartments. When imaging larger structures at low zoom, that should be much less of a problem.
>
> "Also, using a second Ca insensitive dye to construct a "ratio" measurement is unwise -the point of the second wavelength is to control for bleaching and dye distribution etc."
>
> I am also not at all a fan of the approach. It comes with numerous problems, starting with different diffusion rates of the two dyes over different bleaching behaviour, to differential scattering of the two emission wavelengths, leading to a depth-dependent gradient in the ratio, that would need to be corrected for. Having said all that, it has become pretty much standard in neuroscience to report 'green over red' ratios. See publications by the Sabatini for examples.
>
> Best, Chris

Mark  B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[Christian Wilms]

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Hi Mark,

>Any idea what is the cause of the difference between the measurements you cite and the
conclusions of Szmacinski et al (Photochem Photobiol. 1993 Sep;58(3):341-5): "The
emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-
photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and
two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-
1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4
s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. “?

I can only hazard a guess, as it is surprising that the two reported values are so different.

Reading the Szmacinski paper, they seem to have estimated the cross-section by
comparison with a standard. In constrast Xu and Webb
(http://www.pnas.org/content/93/20/10763.long) seem to have measured it directly. But, as
the former estimation is common methodology, I doubt that alone can explain the
difference.

Best, Chris

[De Tombe, Pieter]

Hi,

We used 2P exception to measure radiometric Indo-1 Ca2+ transients in cardiac myocytes; we needed red excitation to prevent breakdown of Blebbistatin, a compound that uncouples
contractile proteins from Ca2+ activation but is photo-inactivated by blue light.
see: Farman GP, Tachampa K, Mateja R, Cazorla O, Lacampagne A, de Tombe PP. Blebbistatin: use as inhibitor of muscle contraction. Pflugers Arch. 2008 Mar;455(6):995–1005.

-----------------------------------------------------------
Pieter P. de Tombe, Ph.D.
James R. DePauw Professor of Physiology
Chair, Department of Cell and Molecular Physiology
Loyola University Chicago
2160 South First Ave.
Stritch School of Medicine
Maywood, IL  60153-5500
[phone]
[phone] (FAX)
[hidden email]

> On Apr 26, 2016, at 5:01 PM, Christian Wilms <[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.
> *****
>
> Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.
>
> Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.
>
> Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.
>
> FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.
>
> An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.
>
> If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)
>
> Good luck with your experiments!
>
> Best, Christian

[Christian Wilms]

*****
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*****

My apologies for the previous message. It seems to have been caught in the system for
nearly 48 hours. Until now that is.

[De Tombe, Pieter]

Oops, auto corrector mishap ... 2P excitation … sorry

P.

> On Apr 28, 2016, at 9:37 AM, De Tombe, Pieter <[hidden email]> wrote:
>
> Hi,
>
> We used 2P exception to measure radiometric Indo-1 Ca2+ transients in cardiac myocytes; we needed red excitation to prevent breakdown of Blebbistatin, a compound that uncouples
> contractile proteins from Ca2+ activation but is photo-inactivated by blue light.
> see: Farman GP, Tachampa K, Mateja R, Cazorla O, Lacampagne A, de Tombe PP. Blebbistatin: use as inhibitor of muscle contraction. Pflugers Arch. 2008 Mar;455(6):995–1005.
>
> -----------------------------------------------------------
> Pieter P. de Tombe, Ph.D.
> James R. DePauw Professor of Physiology
> Chair, Department of Cell and Molecular Physiology
> Loyola University Chicago
> 2160 South First Ave.
> Stritch School of Medicine
> Maywood, IL  60153-5500
> [phone]
> [phone] (FAX)
> [hidden email]
>
>> On Apr 26, 2016, at 5:01 PM, Christian Wilms <[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.
>> *****
>>
>> Ratiometric measurements using two-photon excitation is not easy. It helps to keep that in mind.
>>
>> Fura-2 *might* work if you use a laser that allow fast enough  wavelength switching or use two lasers. But due to the broadened excitation bands for the separate peaks (when compared to one-photon excitation), it is by no means clear that it will be successful.
>>
>> Emission-ratiometric indicators would be the way to go. But the calcium-bound form of Indo-1 has an extremely poor two-photon cross-section, making it unsuited for ratiometric imaging when using 2P-excitation.
>>
>> FRET-based genetically encoded calcium indicators such as YC-2.60 can be used for ratiometric detection using 2P-excitation, but one needs to keep in mind that their calcium binding properties tend to be very non-linear, making calibration difficult.
>>
>> An approach many people have been using, although mostly without the step to actually attempting to quantify the [Ca2+]  is to use a mix of dyes: one calcium indicator (e.g. Fluo-4) and one [Ca2+]-insensitive dye (e.g. Alexa 594). This combination yields ratiometric traces with a very good signal-to-noise ratio, but I would be very sceptical of papers trying to actually claim it reflects an absolute calcium concentration.
>>
>> If actually quantifying calcium is what you want to achieve, there are approaches using single wavelength indicators such as Fluo-4 or OGB-1 that might be worth looking into. If you are working in a system that allows you to saturate the indicator in the cell at the end of an experiment, have a look at Miquel Maravall's 2000 Biophysical Journal paper (DOI: 10.1016/S0006-3495(00)76809-3). And finally, a shameless plug: fluorescence lifetime imaging enables ratiometric analysis of a single wavelength indicator. See Wilms et al. Cell Calcium, 2006 (DOI: 10.1016/j.ceca.2006.03.006)
>>
>> Good luck with your experiments!
>>
>> Best, Christian
>