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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 <[log in to unmask]> 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 [[log in to unmask]]" im
> Auftrag von "Marco Dal Maschio [[log in to unmask]]
> Gesendet: Montag, 12. Dezember 2011 18:22
> Bis: [log in to unmask]
> 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 <[log in to unmask]
> >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:[log in to unmask]]
> > On
> > Behalf Of Andreas Bruckbauer
> > Sent: Monday, December 12, 2011 11:18 AM
> > To: [log in to unmask]
> > 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 <[log in to unmask]>
> >
> > To: CONFOCALMICROSCOPY <[log in to unmask]>
> >
> > 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:[log in to unmask]]
> > On
> >
> >
> >
> > Behalf Of Stéphane Pagès
> >
> >
> >
> > Sent: Monday, 12 December 2011 7:47 PM
> >
> >
> >
> > To: [log in to unmask]
> >
> >
> >
> > 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
>
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