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Two photon fluorescence imaging at GHz repetition rate

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Stéphane Pagès

Two photon fluorescence imaging at GHz repetition rate

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

Hi everybody,
I am considering to buy a new laser to do in vivo two photon imaging.
Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
for that purpose (cheaper than classical TISa at 85 MHz). They claim
that the photodamage is lower because the average power to the sample
is lower.
Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
describe two photon excited fluorescence imaging with such lasers, I
wonder about the photophysical mechanisms involved in excitation of
classical dye (let's say GFP) under these conditions.
The fluorescence life time of GFP (as demonstarted by FLIM and
spectroscopic measurements) is larger than few nanoseconds. And this
is the same thing for the large majority of dyes used in biology.
With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
and therefore the next pulse arrives to the dye while the system is
still in its excited state.
As I understand, there is a risk of either photoionization or at least
destruction of the dye in this context (with creation of singlet
oxygen).
Maybe I am wrong. What is your feeling about this new way to image
biological samples ?

Thanks,
Stéphane

Tobias Rose

AW: Two photon fluorescence imaging at GHz repetition rate

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

This is an excellent question and one I asked myself several times as well. Especially since the opposite is also true: http://goo.gl/NDXzK. Similarly, scanning much faster also reduces photobleaching and -damage (http://goo.gl/PQ4kY), probably again by preventing hitting fluorophores within their fluorescence or more likely absorbing dark-state lifetime. This is a messy situation, indeed.

What would be an experiment to finally settle this? A rigorous measurement of bleachrate and emitted photons per laserpulse (fixed number of pulses) over a wide range of repetition rates (1Mhz -> 2 GHz) and constant power per pulse? Since the pulse-width should also be comparable the only way of doing this would probably be to buy a GHz laser and a pulse picker.

Could someone please do this ;)?

Best
Tobias

________________________________________
Von: Confocal Microscopy List [[hidden email]]" im Auftrag von "Stéphane Pagès [[hidden email]]
Gesendet: Montag, 12. Dezember 2011 09:46
Bis: [hidden email]
Betreff: Two photon fluorescence imaging at GHz repetition rate

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

Hi everybody,
I am considering to buy a new laser to do in vivo two photon imaging.
Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
for that purpose (cheaper than classical TISa at 85 MHz). They claim
that the photodamage is lower because the average power to the sample
is lower.
Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
describe two photon excited fluorescence imaging with such lasers, I
wonder about the photophysical mechanisms involved in excitation of
classical dye (let's say GFP) under these conditions.
The fluorescence life time of GFP (as demonstarted by FLIM and
spectroscopic measurements) is larger than few nanoseconds. And this
is the same thing for the large majority of dyes used in biology.
With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
and therefore the next pulse arrives to the dye while the system is
still in its excited state.
As I understand, there is a risk of either photoionization or at least
destruction of the dye in this context (with creation of singlet
oxygen).
Maybe I am wrong. What is your feeling about this new way to image
biological samples ?

Thanks,
Stéphane

Guy Cox-2

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Stéphane Pagès

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

I think this isn't too much of an issue - remember TWO more photons will need to arrive during the lifetime to have much effect. And in conventional confocal one is illuminating with CW lasers so that extra photons are arriving all the time. The big downside with these high-rep lasers is that they aren't really suitable for FLIM, but if that isn't part of your plans I'd think they would be OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stéphane Pagès
Sent: Monday, 12 December 2011 7:47 PM
To: [hidden email]
Subject: Two photon fluorescence imaging at GHz repetition rate

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

Hi everybody,
I am considering to buy a new laser to do in vivo two photon imaging.
Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
for that purpose (cheaper than classical TISa at 85 MHz). They claim
that the photodamage is lower because the average power to the sample
is lower.
Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
describe two photon excited fluorescence imaging with such lasers, I
wonder about the photophysical mechanisms involved in excitation of
classical dye (let's say GFP) under these conditions.
The fluorescence life time of GFP (as demonstarted by FLIM and
spectroscopic measurements) is larger than few nanoseconds. And this
is the same thing for the large majority of dyes used in biology.
With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
and therefore the next pulse arrives to the dye while the system is
still in its excited state.
As I understand, there is a risk of either photoionization or at least
destruction of the dye in this context (with creation of singlet
oxygen).
Maybe I am wrong. What is your feeling about this new way to image
biological samples ?

Thanks,
Stéphane

Andreas Bruckbauer

Re: Two photon fluorescence imaging at GHz repetition rate

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

Also consider the extreme low probability that two photons are actually absorbed by the dye, that is why you need pulsed lasers in the first place. If you would get one fluorescence photon for every puls of the normal 80 MHz laser for every dye molecule you would get 80 million photons / dye molecule, i would think you can be lucky to get 100

best wishes

Andreas

-----Original Message-----
From: Guy Cox <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 12 Dec 2011 10:49
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

I think this isn't too much of an issue - remember TWO more photons will need to

arrive during the lifetime to have much effect. And in conventional confocal

one is illuminating with CW lasers so that extra photons are arriving all the

time. The big downside with these high-rep lasers is that they aren't really

suitable for FLIM, but if that isn't part of your plans I'd think they would be

OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Stéphane Pagès

Sent: Monday, 12 December 2011 7:47 PM

To: [hidden email]

Subject: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Hi everybody,

I am considering to buy a new laser to do in vivo two photon imaging.

Some companies sell TiSa lasers with a high repetition rates 1-2 GHz

for that purpose (cheaper than classical TISa at 85 MHz). They claim

that the photodamage is lower because the average power to the sample

is lower.

Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)

describe two photon excited fluorescence imaging with such lasers, I

wonder about the photophysical mechanisms involved in excitation of

classical dye (let's say GFP) under these conditions.

The fluorescence life time of GFP (as demonstarted by FLIM and

spectroscopic measurements) is larger than few nanoseconds. And this

is the same thing for the large majority of dyes used in biology.

With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns

and therefore the next pulse arrives to the dye while the system is

still in its excited state.

As I understand, there is a risk of either photoionization or at least

destruction of the dye in this context (with creation of singlet

oxygen).

Maybe I am wrong. What is your feeling about this new way to image

biological samples ?

Thanks,

Stéphane

Vladimir Ghukasyan-2

Re: Two photon fluorescence imaging at GHz repetition rate

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

In addition to all already said, modern software allows analysis of an
incomplete decay. In particular, this is incorporated in commercially
available SPCImage from Becker-Hickl (no commercial interest).

I expect that with GHz lasers you may also have limited depth of
penetration as compared to MHz lasers. It would be interesting to
compare

Best,
Vladimir

=====================
Vladimir Ghukasyan,
Confocal and Multiphoton Imaging Facility
Neuroscience Center
University of North Carolina

On Mon, Dec 12, 2011 at 11:18 AM, Andreas Bruckbauer <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Also consider the extreme low probability that two photons are actually absorbed by the dye, that is why you need pulsed lasers in the first place. If you would get one fluorescence photon for every puls of the normal 80 MHz laser for every dye molecule you would get 80 million photons / dye molecule, i would think you can be lucky to get 100
>
> best wishes
>
> Andreas
>
>
>
>
>
> -----Original Message-----
> From: Guy Cox <[hidden email]>
> To: CONFOCALMICROSCOPY <[hidden email]>
> Sent: Mon, 12 Dec 2011 10:49
> Subject: Re: Two photon fluorescence imaging at GHz repetition rate
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> I think this isn't too much of an issue - remember TWO more photons will need to
>
> arrive during the lifetime to have much effect. And in conventional confocal
>
> one is illuminating with CW lasers so that extra photons are arriving all the
>
> time. The big downside with these high-rep lasers is that they aren't really
>
> suitable for FLIM, but if that isn't part of your plans I'd think they would be
>
> OK. No personal experience, though.
>
>
>
> Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
>
> by Guy Cox CRC Press / Taylor & Francis
>
> http://www.guycox.com/optical.htm
>
> ______________________________________________
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
> Australian Centre for Microscopy & Microanalysis,
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>
> Mobile 0413 281 861
>
> ______________________________________________
>
> http://www.guycox.net
>
>
>
>
>
>
>
> -----Original Message-----
>
> From: Confocal Microscopy List [mailto:[hidden email]] On
>
> Behalf Of Stéphane Pagès
>
> Sent: Monday, 12 December 2011 7:47 PM
>
> To: [hidden email]
>
> Subject: Two photon fluorescence imaging at GHz repetition rate
>
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Hi everybody,
>
> I am considering to buy a new laser to do in vivo two photon imaging.
>
> Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
>
> for that purpose (cheaper than classical TISa at 85 MHz). They claim
>
> that the photodamage is lower because the average power to the sample
>
> is lower.
>
> Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
>
> describe two photon excited fluorescence imaging with such lasers, I
>
> wonder about the photophysical mechanisms involved in excitation of
>
> classical dye (let's say GFP) under these conditions.
>
> The fluorescence life time of GFP (as demonstarted by FLIM and
>
> spectroscopic measurements) is larger than few nanoseconds. And this
>
> is the same thing for the large majority of dyes used in biology.
>
> With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
>
> and therefore the next pulse arrives to the dye while the system is
>
> still in its excited state.
>
> As I understand, there is a risk of either photoionization or at least
>
> destruction of the dye in this context (with creation of singlet
>
> oxygen).
>
> Maybe I am wrong. What is your feeling about this new way to image
>
> biological samples ?
>
>
>
> Thanks,
>
> Stéphane
>
>
>

Tobias Rose

AW: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Andreas Bruckbauer

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

well: but since triplet relaxation lifetime is one order of magnitude slower this could well be an issue (again: Donnert et al., Nature Methods 2007).

________________________________________
Von: Confocal Microscopy List [[hidden email]]" im Auftrag von "Andreas Bruckbauer [[hidden email]]
Gesendet: Montag, 12. Dezember 2011 17:18
Bis: [hidden email]
Betreff: Re: Two photon fluorescence imaging at GHz repetition rate

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

Also consider the extreme low probability that two photons are actually absorbed by the dye, that is why you need pulsed lasers in the first place. If you would get one fluorescence photon for every puls of the normal 80 MHz laser for every dye molecule you would get 80 million photons / dye molecule, i would think you can be lucky to get 100

best wishes

Andreas

-----Original Message-----
From: Guy Cox <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 12 Dec 2011 10:49
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

I think this isn't too much of an issue - remember TWO more photons will need to

arrive during the lifetime to have much effect. And in conventional confocal

one is illuminating with CW lasers so that extra photons are arriving all the

time. The big downside with these high-rep lasers is that they aren't really

suitable for FLIM, but if that isn't part of your plans I'd think they would be

OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Stéphane Pagès

Sent: Monday, 12 December 2011 7:47 PM

To: [hidden email]

Subject: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Hi everybody,

I am considering to buy a new laser to do in vivo two photon imaging.

Some companies sell TiSa lasers with a high repetition rates 1-2 GHz

for that purpose (cheaper than classical TISa at 85 MHz). They claim

that the photodamage is lower because the average power to the sample

is lower.

Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)

describe two photon excited fluorescence imaging with such lasers, I

wonder about the photophysical mechanisms involved in excitation of

classical dye (let's say GFP) under these conditions.

The fluorescence life time of GFP (as demonstarted by FLIM and

spectroscopic measurements) is larger than few nanoseconds. And this

is the same thing for the large majority of dyes used in biology.

With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns

and therefore the next pulse arrives to the dye while the system is

still in its excited state.

As I understand, there is a risk of either photoionization or at least

destruction of the dye in this context (with creation of singlet

oxygen).

Maybe I am wrong. What is your feeling about this new way to image

biological samples ?

Thanks,

Stéphane

pranjal nahar

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Stéphane Pagès

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

Since we are on this subject, is there a publication out there or can
anyone explain the relationship between pulse with and depth penetration in
tissues for Ti:Saph lasers (if all the other variables, NA of the
objective, wavelength, pre-chirping, laser power, PMTs etc. were kept
constant)? Does shorter pulse width equal to deeper penetration in the
tissue and better signal to noise ratios?

Cheers,

Pranjal

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Andreas Bruckbauer
Sent: Monday, December 12, 2011 11:18 AM
To: [hidden email]
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Also consider the extreme low probability that two photons are actually
absorbed by the dye, that is why you need pulsed lasers in the first place.
If you would get one fluorescence photon for every puls of the normal 80
MHz laser for every dye molecule you would get 80 million photons / dye
molecule, i would think you can be lucky to get 100

best wishes

Andreas

-----Original Message-----

From: Guy Cox <[hidden email]>

To: CONFOCALMICROSCOPY <[hidden email]>

Sent: Mon, 12 Dec 2011 10:49

Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

I think this isn't too much of an issue - remember TWO more photons will
need to

arrive during the lifetime to have much effect. And in conventional
confocal

one is illuminating with CW lasers so that extra photons are arriving all
the

time. The big downside with these high-rep lasers is that they aren't
really

suitable for FLIM, but if that isn't part of your plans I'd think they
would be

OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Stéphane Pagès

Sent: Monday, 12 December 2011 7:47 PM

To: [hidden email]

Subject: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Hi everybody,

I am considering to buy a new laser to do in vivo two photon imaging.

Some companies sell TiSa lasers with a high repetition rates 1-2 GHz

for that purpose (cheaper than classical TISa at 85 MHz). They claim

that the photodamage is lower because the average power to the sample

is lower.

Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)

describe two photon excited fluorescence imaging with such lasers, I

wonder about the photophysical mechanisms involved in excitation of

classical dye (let's say GFP) under these conditions.

The fluorescence life time of GFP (as demonstarted by FLIM and

spectroscopic measurements) is larger than few nanoseconds. And this

is the same thing for the large majority of dyes used in biology.

With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns

and therefore the next pulse arrives to the dye while the system is

still in its excited state.

As I understand, there is a risk of either photoionization or at least

destruction of the dye in this context (with creation of singlet

oxygen).

Maybe I am wrong. What is your feeling about this new way to image

biological samples ?

Thanks,

Stéphane

Tobias Rose

AW: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Tobias Rose

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

correction: triplet/dark state lifetime should be on the level of usecs, even... but I did not do any tests either.

If I understand the question of the original poster correctly it was not about lifetime measurements but whether GHz repetition rate is good or bad for 2p microscopy. This puts the mentioned Betzig paper straight against the Hell paper (without contradiction, that is): The first claims that fast rates at low peak power reduce photodamage while maximizing fluorescent yield / time. The second claims that 1Mhz repetition yields the most fluorescence per molecule before it's bleached (due to the prevention of multi-excition within some kind of dark but excitable or triplet state lifetime). TO my understanding one ends up with two alternatives high peak power slow repetition and low peak power high repetition... I cannot make up my mind what would be better.

best,T
________________________________________
Von: Confocal Microscopy List [[hidden email]]" im Auftrag von "Tobias Rose [[hidden email]]
Gesendet: Montag, 12. Dezember 2011 17:50
Bis: [hidden email]
Betreff: AW: Two photon fluorescence imaging at GHz repetition rate

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

well: but since triplet relaxation lifetime is one order of magnitude slower this could well be an issue (again: Donnert et al., Nature Methods 2007).

________________________________________
Von: Confocal Microscopy List [[hidden email]]" im Auftrag von "Andreas Bruckbauer [[hidden email]]
Gesendet: Montag, 12. Dezember 2011 17:18
Bis: [hidden email]
Betreff: Re: Two photon fluorescence imaging at GHz repetition rate

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

Also consider the extreme low probability that two photons are actually absorbed by the dye, that is why you need pulsed lasers in the first place. If you would get one fluorescence photon for every puls of the normal 80 MHz laser for every dye molecule you would get 80 million photons / dye molecule, i would think you can be lucky to get 100

best wishes

Andreas

-----Original Message-----
From: Guy Cox <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Mon, 12 Dec 2011 10:49
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

I think this isn't too much of an issue - remember TWO more photons will need to

arrive during the lifetime to have much effect. And in conventional confocal

one is illuminating with CW lasers so that extra photons are arriving all the

time. The big downside with these high-rep lasers is that they aren't really

suitable for FLIM, but if that isn't part of your plans I'd think they would be

OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Stéphane Pagès

Sent: Monday, 12 December 2011 7:47 PM

To: [hidden email]

Subject: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Hi everybody,

I am considering to buy a new laser to do in vivo two photon imaging.

Some companies sell TiSa lasers with a high repetition rates 1-2 GHz

for that purpose (cheaper than classical TISa at 85 MHz). They claim

that the photodamage is lower because the average power to the sample

is lower.

Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)

describe two photon excited fluorescence imaging with such lasers, I

wonder about the photophysical mechanisms involved in excitation of

classical dye (let's say GFP) under these conditions.

The fluorescence life time of GFP (as demonstarted by FLIM and

spectroscopic measurements) is larger than few nanoseconds. And this

is the same thing for the large majority of dyes used in biology.

With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns

and therefore the next pulse arrives to the dye while the system is

still in its excited state.

As I understand, there is a risk of either photoionization or at least

destruction of the dye in this context (with creation of singlet

oxygen).

Maybe I am wrong. What is your feeling about this new way to image

biological samples ?

Thanks,

Stéphane

Marco Dal Maschio

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by pranjal nahar

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

Just to start the discussion

Two-photon imaging to a depth of 1000 mm in living brains
by use of a Ti:Al2O3 regenerative amplif ier

Patrick Theer, Mazahir T. Hasan, and Winfried Denk

On the fundamental imaging-depth limit in
two-photon microscopy

Patrick Theer* and Winfried Denk

On Mon, Dec 12, 2011 at 6:01 PM, pranjal nahar <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Since we are on this subject, is there a publication out there or can
> anyone explain the relationship between pulse with and depth penetration in
> tissues for Ti:Saph lasers (if all the other variables, NA of the
> objective, wavelength, pre-chirping, laser power, PMTs etc. were kept
> constant)? Does shorter pulse width equal to deeper penetration in the
> tissue and better signal to noise ratios?
>
>
>
>
>
> Cheers,
>
>
>
> Pranjal
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
> Behalf Of Andreas Bruckbauer
> Sent: Monday, December 12, 2011 11:18 AM
> To: [hidden email]
> Subject: Re: Two photon fluorescence imaging at GHz repetition rate
>
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
>
>
> Also consider the extreme low probability that two photons are actually
> absorbed by the dye, that is why you need pulsed lasers in the first place.
> If you would get one fluorescence photon for every puls of the normal 80
> MHz laser for every dye molecule you would get 80 million photons / dye
> molecule, i would think you can be lucky to get 100
>
>
>
> best wishes
>
>
>
> Andreas
>
>
>
>
>
>
>
>
>
>
>
> -----Original Message-----
>
> From: Guy Cox <[hidden email]>
>
> To: CONFOCALMICROSCOPY <[hidden email]>
>
> Sent: Mon, 12 Dec 2011 10:49
>
> Subject: Re: Two photon fluorescence imaging at GHz repetition rate
>
>
>
>
>
> *****
>
>
>
> To join, leave or search the confocal microscopy listserv, go to:
>
>
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>
>
> *****
>
>
>
>
>
>
>
> I think this isn't too much of an issue - remember TWO more photons will
> need to
>
>
>
> arrive during the lifetime to have much effect. And in conventional
> confocal
>
>
>
> one is illuminating with CW lasers so that extra photons are arriving all
> the
>
>
>
> time. The big downside with these high-rep lasers is that they aren't
> really
>
>
>
> suitable for FLIM, but if that isn't part of your plans I'd think they
> would be
>
>
>
> OK. No personal experience, though.
>
>
>
>
>
>
>
> Guy
>
>
>
>
>
>
>
> Optical Imaging Techniques in Cell Biology
>
>
>
> by Guy Cox CRC Press / Taylor & Francis
>
>
>
> http://www.guycox.com/optical.htm
>
>
>
> ______________________________________________
>
>
>
> Associate Professor Guy Cox, MA, DPhil(Oxon)
>
>
>
> Australian Centre for Microscopy & Microanalysis,
>
>
>
> Madsen Building F09, University of Sydney, NSW 2006
>
>
>
>
>
>
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>
>
>
> Mobile 0413 281 861
>
>
>
> ______________________________________________
>
>
>
> http://www.guycox.net
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> -----Original Message-----
>
>
>
> From: Confocal Microscopy List [mailto:[hidden email]]
> On
>
>
>
> Behalf Of Stéphane Pagès
>
>
>
> Sent: Monday, 12 December 2011 7:47 PM
>
>
>
> To: [hidden email]
>
>
>
> Subject: Two photon fluorescence imaging at GHz repetition rate
>
>
>
>
>
>
>
> *****
>
>
>
> To join, leave or search the confocal microscopy listserv, go to:
>
>
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>
>
> *****
>
>
>
>
>
>
>
> Hi everybody,
>
>
>
> I am considering to buy a new laser to do in vivo two photon imaging.
>
>
>
> Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
>
>
>
> for that purpose (cheaper than classical TISa at 85 MHz). They claim
>
>
>
> that the photodamage is lower because the average power to the sample
>
>
>
> is lower.
>
>
>
> Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
>
>
>
> describe two photon excited fluorescence imaging with such lasers, I
>
>
>
> wonder about the photophysical mechanisms involved in excitation of
>
>
>
> classical dye (let's say GFP) under these conditions.
>
>
>
> The fluorescence life time of GFP (as demonstarted by FLIM and
>
>
>
> spectroscopic measurements) is larger than few nanoseconds. And this
>
>
>
> is the same thing for the large majority of dyes used in biology.
>
>
>
> With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
>
>
>
> and therefore the next pulse arrives to the dye while the system is
>
>
>
> still in its excited state.
>
>
>
> As I understand, there is a risk of either photoionization or at least
>
>
>
> destruction of the dye in this context (with creation of singlet
>
>
>
> oxygen).
>
>
>
> Maybe I am wrong. What is your feeling about this new way to image
>
>
>
> biological samples ?
>
>
>
>
>
>
>
> Thanks,
>
>
>
> Stéphane
>

--
Marco Dal Maschio
Italian institute of technology

Chen, De (NIH/NCI) [C]

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by pranjal nahar

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

The fact is the short pulse provides the peak power for two-photon absorption, while the long wavelength of two-photon exciation increases the penetration in tissue.

________________________________________
From: pranjal nahar [[hidden email]]
Sent: Monday, December 12, 2011 12:01 PM
To: [hidden email]
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

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

Since we are on this subject, is there a publication out there or can
anyone explain the relationship between pulse with and depth penetration in
tissues for Ti:Saph lasers (if all the other variables, NA of the
objective, wavelength, pre-chirping, laser power, PMTs etc. were kept
constant)? Does shorter pulse width equal to deeper penetration in the
tissue and better signal to noise ratios?

Cheers,

Pranjal

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Andreas Bruckbauer
Sent: Monday, December 12, 2011 11:18 AM
To: [hidden email]
Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Also consider the extreme low probability that two photons are actually
absorbed by the dye, that is why you need pulsed lasers in the first place.
If you would get one fluorescence photon for every puls of the normal 80
MHz laser for every dye molecule you would get 80 million photons / dye
molecule, i would think you can be lucky to get 100

best wishes

Andreas

-----Original Message-----

From: Guy Cox <[hidden email]>

To: CONFOCALMICROSCOPY <[hidden email]>

Sent: Mon, 12 Dec 2011 10:49

Subject: Re: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

I think this isn't too much of an issue - remember TWO more photons will
need to

arrive during the lifetime to have much effect. And in conventional
confocal

one is illuminating with CW lasers so that extra photons are arriving all
the

time. The big downside with these high-rep lasers is that they aren't
really

suitable for FLIM, but if that isn't part of your plans I'd think they
would be

OK. No personal experience, though.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

-----Original Message-----

From: Confocal Microscopy List [mailto:[hidden email]] On

Behalf Of Stéphane Pagès

Sent: Monday, 12 December 2011 7:47 PM

To: [hidden email]

Subject: Two photon fluorescence imaging at GHz repetition rate

*****

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

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

*****

Hi everybody,

I am considering to buy a new laser to do in vivo two photon imaging.

Some companies sell TiSa lasers with a high repetition rates 1-2 GHz

for that purpose (cheaper than classical TISa at 85 MHz). They claim

that the photodamage is lower because the average power to the sample

is lower.

Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)

describe two photon excited fluorescence imaging with such lasers, I

wonder about the photophysical mechanisms involved in excitation of

classical dye (let's say GFP) under these conditions.

The fluorescence life time of GFP (as demonstarted by FLIM and

spectroscopic measurements) is larger than few nanoseconds. And this

is the same thing for the large majority of dyes used in biology.

With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns

and therefore the next pulse arrives to the dye while the system is

still in its excited state.

As I understand, there is a risk of either photoionization or at least

destruction of the dye in this context (with creation of singlet

oxygen).

Maybe I am wrong. What is your feeling about this new way to image

biological samples ?

Thanks,

Stéphane

Tobias Rose

AW: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Marco Dal Maschio

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

These are excellent papers and also good example for crazy high peak power and very low repetition rate...
Does anyone know if anyone ever did chronic (repeated) imaging using this "RAMM" [regenerative amplification multiphoton microscopy] technique http://www.nature.com/neuro/journal/v14/n8/full/nn.2879.html?

It always sounded a bit harsh to me...

________________________________________
Von: Confocal Microscopy List [[hidden email]]" im Auftrag von "Marco Dal Maschio [[hidden email]]
Gesendet: Montag, 12. Dezember 2011 18:22
Bis: [hidden email]
Betreff: Re: Two photon fluorescence imaging at GHz repetition rate

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

Just to start the discussion

Two-photon imaging to a depth of 1000 mm in living brains
by use of a Ti:Al2O3 regenerative amplif ier

Patrick Theer, Mazahir T. Hasan, and Winfried Denk

On the fundamental imaging-depth limit in
two-photon microscopy

Patrick Theer* and Winfried Denk

On Mon, Dec 12, 2011 at 6:01 PM, pranjal nahar <[hidden email]>wrote:

--
Marco Dal Maschio
Italian institute of technology

Craig Brideau

Re: Two photon fluorescence imaging at GHz repetition rate

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

Our lab went through this same question a year ago. We were going to
purchase an Idesta Ti:Saph and we had the option to get 1GHz or 85MHz as
the repetition rate. We went with 85MHz for two reasons:

1) Non-linear effects such as 2 photon are PEAK energy dependent, so you
have more efficient excitation (photons produces vs. power in) when you use
higher peak energy.
2) The lower repetition rate gives a bit of time for any flurophores to
relax between pulses. In theory this leads to less photobleaching since
the dye has to be in the excited state and then be hit again in order for
photochemical bleaching to occur. I don't buy into the 'less
photobleaching' claims of the GHz lasers for this reason. I think overall
the GHz pulses just excite everything less, so sure you get less
photobleaching but also proportionally lower signal. What you should care
about is bleaching occurring vs signal produced rather than just net lower
photobleaching. After all, if you are producing too much signal you can
always turn the laser down or chirp the pulses out to get lower peak energy.

The one thing I could see a GHz laser being very good for is high-speed
video rate imaging. You'd have lots of pulses per pixel so it would help
with noise. The question would be if the lower peak energy would give you
enough signal at video rate.

Craig

On Mon, Dec 12, 2011 at 11:01 AM, Tobias Rose <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> These are excellent papers and also good example for crazy high peak power
> and very low repetition rate...
> Does anyone know if anyone ever did chronic (repeated) imaging using this
> "RAMM" [regenerative amplification multiphoton microscopy] technique
> http://www.nature.com/neuro/journal/v14/n8/full/nn.2879.html?
>
> It always sounded a bit harsh to me...
>
> ________________________________________
> Von: Confocal Microscopy List [[hidden email]]" im
> Auftrag von "Marco Dal Maschio [[hidden email]]
> Gesendet: Montag, 12. Dezember 2011 18:22
> Bis: [hidden email]
> Betreff: Re: Two photon fluorescence imaging at GHz repetition rate
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Just to start the discussion
>
>
> Two-photon imaging to a depth of 1000 mm in living brains
> by use of a Ti:Al2O3 regenerative amplif ier
>
> Patrick Theer, Mazahir T. Hasan, and Winfried Denk
>
> On the fundamental imaging-depth limit in
> two-photon microscopy
>
> Patrick Theer* and Winfried Denk
>
>
>
>
> On Mon, Dec 12, 2011 at 6:01 PM, pranjal nahar <[hidden email]
> >wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Since we are on this subject, is there a publication out there or can
> > anyone explain the relationship between pulse with and depth penetration
> in
> > tissues for Ti:Saph lasers (if all the other variables, NA of the
> > objective, wavelength, pre-chirping, laser power, PMTs etc. were kept
> > constant)? Does shorter pulse width equal to deeper penetration in the
> > tissue and better signal to noise ratios?
> >
> >
> >
> >
> >
> > Cheers,
> >
> >
> >
> > Pranjal
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On
> > Behalf Of Andreas Bruckbauer
> > Sent: Monday, December 12, 2011 11:18 AM
> > To: [hidden email]
> > Subject: Re: Two photon fluorescence imaging at GHz repetition rate
> >
> >
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> > *****
> >
> >
> >
> >
> >
> > Also consider the extreme low probability that two photons are actually
> > absorbed by the dye, that is why you need pulsed lasers in the first
> place.
> > If you would get one fluorescence photon for every puls of the normal 80
> > MHz laser for every dye molecule you would get 80 million photons / dye
> > molecule, i would think you can be lucky to get 100
> >
> >
> >
> > best wishes
> >
> >
> >
> > Andreas
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> > -----Original Message-----
> >
> > From: Guy Cox <[hidden email]>
> >
> > To: CONFOCALMICROSCOPY <[hidden email]>
> >
> > Sent: Mon, 12 Dec 2011 10:49
> >
> > Subject: Re: Two photon fluorescence imaging at GHz repetition rate
> >
> >
> >
> >
> >
> > *****
> >
> >
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> >
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> >
> >
> > *****
> >
> >
> >
> >
> >
> >
> >
> > I think this isn't too much of an issue - remember TWO more photons will
> > need to
> >
> >
> >
> > arrive during the lifetime to have much effect. And in conventional
> > confocal
> >
> >
> >
> > one is illuminating with CW lasers so that extra photons are arriving all
> > the
> >
> >
> >
> > time. The big downside with these high-rep lasers is that they aren't
> > really
> >
> >
> >
> > suitable for FLIM, but if that isn't part of your plans I'd think they
> > would be
> >
> >
> >
> > OK. No personal experience, though.
> >
> >
> >
> >
> >
> >
> >
> > Guy
> >
> >
> >
> >
> >
> >
> >
> > Optical Imaging Techniques in Cell Biology
> >
> >
> >
> > by Guy Cox CRC Press / Taylor & Francis
> >
> >
> >
> > http://www.guycox.com/optical.htm
> >
> >
> >
> > ______________________________________________
> >
> >
> >
> > Associate Professor Guy Cox, MA, DPhil(Oxon)
> >
> >
> >
> > Australian Centre for Microscopy & Microanalysis,
> >
> >
> >
> > Madsen Building F09, University of Sydney, NSW 2006
> >
> >
> >
> >
> >
> >
> >
> > Phone +61 2 9351 3176 Fax +61 2 9351 7682
> >
> >
> >
> > Mobile 0413 281 861
> >
> >
> >
> > ______________________________________________
> >
> >
> >
> > http://www.guycox.net
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> >
> > -----Original Message-----
> >
> >
> >
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On
> >
> >
> >
> > Behalf Of Stéphane Pagès
> >
> >
> >
> > Sent: Monday, 12 December 2011 7:47 PM
> >
> >
> >
> > To: [hidden email]
> >
> >
> >
> > Subject: Two photon fluorescence imaging at GHz repetition rate
> >
> >
> >
> >
> >
> >
> >
> > *****
> >
> >
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> >
> >
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> >
> >
> > *****
> >
> >
> >
> >
> >
> >
> >
> > Hi everybody,
> >
> >
> >
> > I am considering to buy a new laser to do in vivo two photon imaging.
> >
> >
> >
> > Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
> >
> >
> >
> > for that purpose (cheaper than classical TISa at 85 MHz). They claim
> >
> >
> >
> > that the photodamage is lower because the average power to the sample
> >
> >
> >
> > is lower.
> >
> >
> >
> > Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
> >
> >
> >
> > describe two photon excited fluorescence imaging with such lasers, I
> >
> >
> >
> > wonder about the photophysical mechanisms involved in excitation of
> >
> >
> >
> > classical dye (let's say GFP) under these conditions.
> >
> >
> >
> > The fluorescence life time of GFP (as demonstarted by FLIM and
> >
> >
> >
> > spectroscopic measurements) is larger than few nanoseconds. And this
> >
> >
> >
> > is the same thing for the large majority of dyes used in biology.
> >
> >
> >
> > With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
> >
> >
> >
> > and therefore the next pulse arrives to the dye while the system is
> >
> >
> >
> > still in its excited state.
> >
> >
> >
> > As I understand, there is a risk of either photoionization or at least
> >
> >
> >
> > destruction of the dye in this context (with creation of singlet
> >
> >
> >
> > oxygen).
> >
> >
> >
> > Maybe I am wrong. What is your feeling about this new way to image
> >
> >
> >
> > biological samples ?
> >
> >
> >
> >
> >
> >
> >
> > Thanks,
> >
> >
> >
> > Stéphane
> >
>
>
>
> --
> Marco Dal Maschio
> Italian institute of technology
>

Wolfgang Staroske

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Stéphane Pagès

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

Hi all,

I think Guys statement is not correct, for that case. As the original
poster mentioned the dye is still in the first excited state (S1) after
1ns. So the next energy level to reach is S2. The bandgap between S1 and
S2 is usually much smaller compared to the bandgap from ground state
(S0) to S1. This means that maybe a SINGLE infrared photon is enough to
drive your dye into S1 and further on, leading to ionisation and
bleaching, and you will have tons of these single infrared photons.

If this happens depends on the bandgap between S1 and S2, the problem
is, that it is very hard to measure this bandgap and therefore they are
usually unknown for all dyes.

For 80MHz that doesn't play a role, as the next pulse comes after 12 ns
and therefore no dye is in S1 anymore, only Triplett state and Ground
state are populated. So direct ONE photon absorption can only take place
in the Triplett state.

Bye Wolfgang

Am 20:59, schrieb Guy Cox:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy
> *****
>
> I think this isn't too much of an issue - remember TWO more photons
> will need to arrive during the lifetime to have much effect. And in
> conventional confocal one is illuminating with CW lasers so that extra
> photons are arriving all the time. The big downside with these
> high-rep lasers is that they aren't really suitable for FLIM, but if
> that isn't part of your plans I'd think they would be OK. No personal
> experience, though.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Stéphane Pagès
> Sent: Monday, 12 December 2011 7:47 PM
> To: [hidden email]
> Subject: Two photon fluorescence imaging at GHz repetition rate
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy
> *****
>
> Hi everybody,
> I am considering to buy a new laser to do in vivo two photon imaging.
> Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
> for that purpose (cheaper than classical TISa at 85 MHz). They claim
> that the photodamage is lower because the average power to the sample
> is lower.
> Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
> describe two photon excited fluorescence imaging with such lasers, I
> wonder about the photophysical mechanisms involved in excitation of
> classical dye (let's say GFP) under these conditions.
> The fluorescence life time of GFP (as demonstarted by FLIM and
> spectroscopic measurements) is larger than few nanoseconds. And this
> is the same thing for the large majority of dyes used in biology.
> With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
> and therefore the next pulse arrives to the dye while the system is
> still in its excited state.
> As I understand, there is a risk of either photoionization or at least
> destruction of the dye in this context (with creation of singlet
> oxygen).
> Maybe I am wrong. What is your feeling about this new way to image
> biological samples ?
>
> Thanks,
> Stéphane
>

--
Dr. Wolfgang Staroske

Single Molecule Specialist
Light Microscopy Facility

Technische Universität Dresden
Biotechnology Center
Tatzberg 47/49
01307 Dresden, Germany

Tel.: +49 (0) 351 463-40316
Fax.: +49 (0) 351 463-40342
E-Mail: [hidden email]
Webpage: www.biotec.tu-dresden.de

Craig Brideau

Re: Two photon fluorescence imaging at GHz repetition rate

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

We actually have some sources that are only 1 MHz repetition rate to give
the sample additional recovery time. The connected scopes are still in
progress so I will have to let you know how it works out in a few months.

Craig

On Tue, Dec 13, 2011 at 1:02 AM, Wolfgang Staroske <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hi all,
>
> I think Guys statement is not correct, for that case. As the original
> poster mentioned the dye is still in the first excited state (S1) after
> 1ns. So the next energy level to reach is S2. The bandgap between S1 and S2
> is usually much smaller compared to the bandgap from ground state (S0) to
> S1. This means that maybe a SINGLE infrared photon is enough to drive your
> dye into S1 and further on, leading to ionisation and bleaching, and you
> will have tons of these single infrared photons.
>
> If this happens depends on the bandgap between S1 and S2, the problem is,
> that it is very hard to measure this bandgap and therefore they are usually
> unknown for all dyes.
>
> For 80MHz that doesn't play a role, as the next pulse comes after 12 ns
> and therefore no dye is in S1 anymore, only Triplett state and Ground state
> are populated. So direct ONE photon absorption can only take place in the
> Triplett state.
>
> Bye Wolfgang
>
> Am 20:59, schrieb Guy Cox:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=nfocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy>
>>
>> *****
>>
>> I think this isn't too much of an issue - remember TWO more photons will
>> need to arrive during the lifetime to have much effect. And in
>> conventional confocal one is illuminating with CW lasers so that extra
>> photons are arriving all the time. The big downside with these high-rep
>> lasers is that they aren't really suitable for FLIM, but if that isn't part
>> of your plans I'd think they would be OK. No personal experience, though.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor & Francis
>>
>> http://www.guycox.com/optical.**htm<http://www.guycox.com/optical.htm>
>> ______________________________**________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>>
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________**________________
>> http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU<[hidden email]>]
>> On Behalf Of Stéphane Pagès
>> Sent: Monday, 12 December 2011 7:47 PM
>> To: [hidden email].**EDU <[hidden email]>
>> Subject: Two photon fluorescence imaging at GHz repetition rate
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=nfocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy>
>>
>> *****
>>
>> Hi everybody,
>> I am considering to buy a new laser to do in vivo two photon imaging.
>> Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
>> for that purpose (cheaper than classical TISa at 85 MHz). They claim
>> that the photodamage is lower because the average power to the sample
>> is lower.
>> Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
>> describe two photon excited fluorescence imaging with such lasers, I
>> wonder about the photophysical mechanisms involved in excitation of
>> classical dye (let's say GFP) under these conditions.
>> The fluorescence life time of GFP (as demonstarted by FLIM and
>> spectroscopic measurements) is larger than few nanoseconds. And this
>> is the same thing for the large majority of dyes used in biology.
>> With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
>> and therefore the next pulse arrives to the dye while the system is
>> still in its excited state.
>> As I understand, there is a risk of either photoionization or at least
>> destruction of the dye in this context (with creation of singlet
>> oxygen).
>> Maybe I am wrong. What is your feeling about this new way to image
>> biological samples ?
>>
>> Thanks,
>> Stéphane
>>
>>
> --
> Dr. Wolfgang Staroske
>
> Single Molecule Specialist
> Light Microscopy Facility
>
> Technische Universität Dresden
> Biotechnology Center
> Tatzberg 47/49
> 01307 Dresden, Germany
>
> Tel.: +49 (0) 351 463-40316
> Fax.: +49 (0) 351 463-40342
> E-Mail: [hidden email]-**dresden.de<[hidden email]>
> Webpage: www.biotec.tu-dresden.de
>

Andrew Ridsdale

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Craig Brideau

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

Hello to the list,

I just started listening again after a long absence and find I already
want to make some comments about considering a high rep-rate laser for
in vivo two photon excitation -

The reference from the original post (Ji et al
http://dx.doi.org/10.1038/nmeth.1175) deals with the benefits of
increasing laser repetition rate if the damage mechanisms are of higher
order non-linearities than the process you are trying to excite.

The signal scaling with for TPEF is ~ 1/sqrt(repetition rate*pulse
length). That is: increasing rep rate or pulse length by a factor "X"
requires a compensating average-power increase of sqrt(X) for the same
signal. (Contrary the claim of lower average power the GHz laser
manufacturer).

Note: this would not apply to thick tissues where scattering losses
almost certainly require the highest practical peak-power.

Most of the discussion has focused on the problems of bleaching of a dye
and its relation to excited-state dynamics. However stress/damage to
the sample is often limiting in live cell observation. Of course this
is a messy area, mostly because: 1. viabilities are difficult to define
(or measure) and depend strongly on types of dyes etc, 2. the varying
extent to which it matters for different experiments.

In my opinion, evidence of greater than 2nd-order nonlinear processes
usually being important for damage is fairly strong. Maybe some of the
"cleanest" measurements of power-scaling is looking at thresholds for
cutting in low-pulse-energy regime: often less than 1 nJ - or 80 mW at
80 MHz, and so overlapping with conditions commonly used in TPEF
imaging. Some measurements show scaling powers >5
(http://www.opticsinfobase.org/abstract.cfm?uri=boe-1-2-587&seq=0 ).
The ablation is thought to be due creation of low-density plasmas (Vogel
et al - reference 10 in above: this argument is expanded in the
discussion). If we assume that stress in cells would be occurring
before ablation was observed, we could anticipate that it would follow
similar scaling.

The limit on increasing average power, of course, is the linear
absorption (heating). In most applications there is still some room
before this becomes a problem. Heating is treated in the section on
TPEF microscopy in Pawley's book. A transient temperature increase
depends on many factors including scan speed. I don't have that just
now, or could do a quick estimate for heating from the ~3.5X average
power increase needed to go from 80MHz to 1GHz.

In the last couple years here we've purposely chirped our pulse from
~300fs to ~900fs at the objective focus, and so need to increase our
average power by something around a factor ~1.7X. Our experience has
been (without having hard data to back it up) that there is
significantly improved viability of tissue culture cells in our set up
for extended observation (e.g. ~500+ images), both stained and unstained
(we mostly do CARS). (One good sign of stress in tissue culture cells
is the "cringe" effect where they round up slightly).

I suspect the reason for this are higher-than-2nd-order components in
various damage mechanisms. Again, the weight of these will depend
strongly on conditions, but will almost always be present.

Andy

On Mon, 2011-12-12 at 16:32 -0500, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Our lab went through this same question a year ago. We were going to
> purchase an Idesta Ti:Saph and we had the option to get 1GHz or 85MHz as
> the repetition rate. We went with 85MHz for two reasons:
>
> 1) Non-linear effects such as 2 photon are PEAK energy dependent, so you
> have more efficient excitation (photons produces vs. power in) when you use
> higher peak energy.
> 2) The lower repetition rate gives a bit of time for any flurophores to
> relax between pulses. In theory this leads to less photobleaching since
> the dye has to be in the excited state and then be hit again in order for
> photochemical bleaching to occur. I don't buy into the 'less
> photobleaching' claims of the GHz lasers for this reason. I think overall
> the GHz pulses just excite everything less, so sure you get less
> photobleaching but also proportionally lower signal. What you should care
> about is bleaching occurring vs signal produced rather than just net lower
> photobleaching. After all, if you are producing too much signal you can
> always turn the laser down or chirp the pulses out to get lower peak energy.
>
> The one thing I could see a GHz laser being very good for is high-speed
> video rate imaging. You'd have lots of pulses per pixel so it would help
> with noise. The question would be if the lower peak energy would give you
> enough signal at video rate.
>
> Craig
>
>
> On Mon, Dec 12, 2011 at 11:01 AM, Tobias Rose <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > These are excellent papers and also good example for crazy high peak power
> > and very low repetition rate...
> > Does anyone know if anyone ever did chronic (repeated) imaging using this
> > "RAMM" [regenerative amplification multiphoton microscopy] technique
> > http://www.nature.com/neuro/journal/v14/n8/full/nn.2879.html?
> >
> > It always sounded a bit harsh to me...
> >
> > ________________________________________
> > Von: Confocal Microscopy List [[hidden email]]" im
> > Auftrag von "Marco Dal Maschio [[hidden email]]
> > Gesendet: Montag, 12. Dezember 2011 18:22
> > Bis: [hidden email]
> > Betreff: Re: Two photon fluorescence imaging at GHz repetition rate
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Just to start the discussion
> >
> >
> > Two-photon imaging to a depth of 1000 mm in living brains
> > by use of a Ti:Al2O3 regenerative amplif ier
> >
> > Patrick Theer, Mazahir T. Hasan, and Winfried Denk
> >
> > On the fundamental imaging-depth limit in
> > two-photon microscopy
> >
> > Patrick Theer* and Winfried Denk
> >
> >
> >
> >
> > On Mon, Dec 12, 2011 at 6:01 PM, pranjal nahar <[hidden email]
> > >wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Since we are on this subject, is there a publication out there or can
> > > anyone explain the relationship between pulse with and depth penetration
> > in
> > > tissues for Ti:Saph lasers (if all the other variables, NA of the
> > > objective, wavelength, pre-chirping, laser power, PMTs etc. were kept
> > > constant)? Does shorter pulse width equal to deeper penetration in the
> > > tissue and better signal to noise ratios?
> > >
> > >
> > >
> > >
> > >
> > > Cheers,
> > >
> > >
> > >
> > > Pranjal
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:[hidden email]]
> > > On
> > > Behalf Of Andreas Bruckbauer
> > > Sent: Monday, December 12, 2011 11:18 AM
> > > To: [hidden email]
> > > Subject: Re: Two photon fluorescence imaging at GHz repetition rate
> > >
> > >
> > >
> > > *****
> > >
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >
> > > *****
> > >
> > >
> > >
> > >
> > >
> > > Also consider the extreme low probability that two photons are actually
> > > absorbed by the dye, that is why you need pulsed lasers in the first
> > place.
> > > If you would get one fluorescence photon for every puls of the normal 80
> > > MHz laser for every dye molecule you would get 80 million photons / dye
> > > molecule, i would think you can be lucky to get 100
> > >
> > >
> > >
> > > best wishes
> > >
> > >
> > >
> > > Andreas
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > -----Original Message-----
> > >
> > > From: Guy Cox <[hidden email]>
> > >
> > > To: CONFOCALMICROSCOPY <[hidden email]>
> > >
> > > Sent: Mon, 12 Dec 2011 10:49
> > >
> > > Subject: Re: Two photon fluorescence imaging at GHz repetition rate
> > >
> > >
> > >
> > >
> > >
> > > *****
> > >
> > >
> > >
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > >
> > >
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >
> > >
> > >
> > > *****
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > I think this isn't too much of an issue - remember TWO more photons will
> > > need to
> > >
> > >
> > >
> > > arrive during the lifetime to have much effect. And in conventional
> > > confocal
> > >
> > >
> > >
> > > one is illuminating with CW lasers so that extra photons are arriving all
> > > the
> > >
> > >
> > >
> > > time. The big downside with these high-rep lasers is that they aren't
> > > really
> > >
> > >
> > >
> > > suitable for FLIM, but if that isn't part of your plans I'd think they
> > > would be
> > >
> > >
> > >
> > > OK. No personal experience, though.
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > Guy
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > Optical Imaging Techniques in Cell Biology
> > >
> > >
> > >
> > > by Guy Cox CRC Press / Taylor & Francis
> > >
> > >
> > >
> > > http://www.guycox.com/optical.htm
> > >
> > >
> > >
> > > ______________________________________________
> > >
> > >
> > >
> > > Associate Professor Guy Cox, MA, DPhil(Oxon)
> > >
> > >
> > >
> > > Australian Centre for Microscopy & Microanalysis,
> > >
> > >
> > >
> > > Madsen Building F09, University of Sydney, NSW 2006
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > Phone +61 2 9351 3176 Fax +61 2 9351 7682
> > >
> > >
> > >
> > > Mobile 0413 281 861
> > >
> > >
> > >
> > > ______________________________________________
> > >
> > >
> > >
> > > http://www.guycox.net
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > -----Original Message-----
> > >
> > >
> > >
> > > From: Confocal Microscopy List [mailto:[hidden email]]
> > > On
> > >
> > >
> > >
> > > Behalf Of Stéphane Pagès
> > >
> > >
> > >
> > > Sent: Monday, 12 December 2011 7:47 PM
> > >
> > >
> > >
> > > To: [hidden email]
> > >
> > >
> > >
> > > Subject: Two photon fluorescence imaging at GHz repetition rate
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > *****
> > >
> > >
> > >
> > > To join, leave or search the confocal microscopy listserv, go to:
> > >
> > >
> > >
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >
> > >
> > >
> > > *****
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > Hi everybody,
> > >
> > >
> > >
> > > I am considering to buy a new laser to do in vivo two photon imaging.
> > >
> > >
> > >
> > > Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
> > >
> > >
> > >
> > > for that purpose (cheaper than classical TISa at 85 MHz). They claim
> > >
> > >
> > >
> > > that the photodamage is lower because the average power to the sample
> > >
> > >
> > >
> > > is lower.
> > >
> > >
> > >
> > > Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
> > >
> > >
> > >
> > > describe two photon excited fluorescence imaging with such lasers, I
> > >
> > >
> > >
> > > wonder about the photophysical mechanisms involved in excitation of
> > >
> > >
> > >
> > > classical dye (let's say GFP) under these conditions.
> > >
> > >
> > >
> > > The fluorescence life time of GFP (as demonstarted by FLIM and
> > >
> > >
> > >
> > > spectroscopic measurements) is larger than few nanoseconds. And this
> > >
> > >
> > >
> > > is the same thing for the large majority of dyes used in biology.
> > >
> > >
> > >
> > > With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
> > >
> > >
> > >
> > > and therefore the next pulse arrives to the dye while the system is
> > >
> > >
> > >
> > > still in its excited state.
> > >
> > >
> > >
> > > As I understand, there is a risk of either photoionization or at least
> > >
> > >
> > >
> > > destruction of the dye in this context (with creation of singlet
> > >
> > >
> > >
> > > oxygen).
> > >
> > >
> > >
> > > Maybe I am wrong. What is your feeling about this new way to image
> > >
> > >
> > >
> > > biological samples ?
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > Thanks,
> > >
> > >
> > >
> > > Stéphane
> > >
> >
> >
> >
> > --
> > Marco Dal Maschio
> > Italian institute of technology
> >

--
Andrew Ridsdale
Steacie Institute for Molecular Sciences
National Research Council of Canada
100 Sussex Drive
Ottawa ON K1A 0R6
Canada

Steffen Dietzel

Re: Two photon fluorescence imaging at GHz repetition rate

In reply to this post by Wolfgang Staroske

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

Hi everybody,

if Wolfgang is correct, by the same line of reasoning, shouldn't we even
be in trouble within one pulse of a MHz Laser? To excite the dye to S1,
two photons have to arrive within attosecond range. If this happens
let's say before pulse mid-time of the femtosecond-pulse, there then
should still be plenty of time and photons left to drive the electron up
to S2. Or not?

Steffen

On 13.12.2011 09:02, Wolfgang Staroske wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> I think Guys statement is not correct, for that case. As the original
> poster mentioned the dye is still in the first excited state (S1) after
> 1ns. So the next energy level to reach is S2. The bandgap between S1 and
> S2 is usually much smaller compared to the bandgap from ground state
> (S0) to S1. This means that maybe a SINGLE infrared photon is enough to
> drive your dye into S1 and further on, leading to ionisation and
> bleaching, and you will have tons of these single infrared photons.
>
> If this happens depends on the bandgap between S1 and S2, the problem
> is, that it is very hard to measure this bandgap and therefore they are
> usually unknown for all dyes.
>
> For 80MHz that doesn't play a role, as the next pulse comes after 12 ns
> and therefore no dye is in S1 anymore, only Triplett state and Ground
> state are populated. So direct ONE photon absorption can only take place
> in the Triplett state.
>
> Bye Wolfgang
>
> Am 20:59, schrieb Guy Cox:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy
>> *****
>>
>> I think this isn't too much of an issue - remember TWO more photons
>> will need to arrive during the lifetime to have much effect. And in
>> conventional confocal one is illuminating with CW lasers so that extra
>> photons are arriving all the time. The big downside with these
>> high-rep lasers is that they aren't really suitable for FLIM, but if
>> that isn't part of your plans I'd think they would be OK. No personal
>> experience, though.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor & Francis
>> http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________________________
>> http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Stéphane Pagès
>> Sent: Monday, 12 December 2011 7:47 PM
>> To: [hidden email]
>> Subject: Two photon fluorescence imaging at GHz repetition rate
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=nfocalmicroscopy
>> *****
>>
>> Hi everybody,
>> I am considering to buy a new laser to do in vivo two photon imaging.
>> Some companies sell TiSa lasers with a high repetition rates 1-2 GHz
>> for that purpose (cheaper than classical TISa at 85 MHz). They claim
>> that the photodamage is lower because the average power to the sample
>> is lower.
>> Althought some papers (JI et al. Nat. Methods, 2008 5:197-202)
>> describe two photon excited fluorescence imaging with such lasers, I
>> wonder about the photophysical mechanisms involved in excitation of
>> classical dye (let's say GFP) under these conditions.
>> The fluorescence life time of GFP (as demonstarted by FLIM and
>> spectroscopic measurements) is larger than few nanoseconds. And this
>> is the same thing for the large majority of dyes used in biology.
>> With a repetition rate of 1 GHz, one pulse comes to the dye each 1 ns
>> and therefore the next pulse arrives to the dye while the system is
>> still in its excited state.
>> As I understand, there is a risk of either photoionization or at least
>> destruction of the dye in this context (with creation of singlet
>> oxygen).
>> Maybe I am wrong. What is your feeling about this new way to image
>> biological samples ?
>>
>> Thanks,
>> Stéphane
>>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27, München-Großhadern