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FCS + two photon?

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Edrun Andrea Schnell

FCS + two photon?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi!

I have a short question that I hope you can answer for me. Are there any
commercially available systems on the market today for fluorescence
correlation spectroscopy with two photon excitation? We have the Zeiss
ConfoCor2 today, and I know that Zeiss don't offer this opportunity. We do
not want to buy this, I just need to know if it exists or not.

Best regards,
Edrun Andrea Schnell

--

Edrun Andrea Schnell
Divisional engineer,
Dept. of Physics, NTNU
Hogskoleringen 5, 7491 Trondheim
Norway

Csucs Gabor

Re: FCS + two photon?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Edrun,

Although Zeiss officially doesn't support it, but I know that with the
ConfoCor 3 system you can do also 2photon FCS. Actually it should be
possible also with the Leica setup (given that you buy the necessary
filter sets..).

Cheers Gabor

--
Gabor Csucs
Light Microscopy Centre, ETH Zurich
Schafmattstrasse 18, HPM F16
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone: +41 44 633 6221
Fax: +41 44 632 1298
e-mail: [hidden email]

Craig Brideau

Re: FCS + two photon?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Other than a bit of filter tweaking depending on your dye, it just
boils down to your detectors. If your system is fiber coupled or has
any easy method for switching detectors then it is extremely simple to
set up. If you have access to your PMT signals, and it is of a type
suitable for counting work (fast response, quick recover), just attach
an amplifier-discriminator module to it and count away. Otherwise you
might need to get both a PMT and amp-discriminator, but if your system
is fiber-coupled then it is simple to pipe your signal into the new
PMT via the fiber.

Craig

On Dec 6, 2007 8:19 AM, Csucs Gabor <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Edrun,
>
> Although Zeiss officially doesn't support it, but I know that with the
> ConfoCor 3 system you can do also 2photon FCS. Actually it should be
> possible also with the Leica setup (given that you buy the necessary
> filter sets..).
>
> Cheers Gabor
>
> --
> Gabor Csucs
> Light Microscopy Centre, ETH Zurich
> Schafmattstrasse 18, HPM F16
> CH-8093, Zurich, Switzerland
>
> Web: www.lmc.ethz.ch
> Phone: +41 44 633 6221
> Fax: +41 44 632 1298
> e-mail: [hidden email]
>

Holly L. AARON

Re: FCS + two photon?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi, Edrun -

You can do FCS with 2-photon excitation using the Becker-Hickl SPC-830 card.
We are new to this card, which provides the opportunity to do FCS for us for
the first time, so are still at the beginning of the learning curve. We are
using it with a Zeiss 510 NLO.

-Holly
__________________
Holly L. Aaron
CRL Molecular Imaging Center
http://imaging.berkeley.edu

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Craig Brideau
Sent: Thursday, December 06, 2007 10:36 AM
To: [hidden email]
Subject: Re: FCS + two photon?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Other than a bit of filter tweaking depending on your dye, it just
boils down to your detectors. If your system is fiber coupled or has
any easy method for switching detectors then it is extremely simple to
set up. If you have access to your PMT signals, and it is of a type
suitable for counting work (fast response, quick recover), just attach
an amplifier-discriminator module to it and count away. Otherwise you
might need to get both a PMT and amp-discriminator, but if your system
is fiber-coupled then it is simple to pipe your signal into the new
PMT via the fiber.

Craig

On Dec 6, 2007 8:19 AM, Csucs Gabor <[hidden email]> wrote:

B. Prabhakar Pandian

Microscope enclosure chambers

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello,
We are looking for a microscope enclosure chambers that will
control temperature, humidity and gas with feedback for long term studies.
I am looking for enclosures that will also allow ports for tubing to
come out of the chamber. The tubing is connected to a syringe pump
outside the enclosure.

Any suggestions with feedback on companies to checkout will be great.

Thanks,

-Prabhakar

Craig Brideau

Re: Microscope enclosure chambers

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

When you say gas, are you filling the enclosure with an inert gas or
some other gas? Or do you just want to restrict air flow around the
microscope?

Craig

On Dec 6, 2007 2:59 PM, B. Prabhakar Pandian <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello,
> We are looking for a microscope enclosure chambers that will
> control temperature, humidity and gas with feedback for long term studies.
> I am looking for enclosures that will also allow ports for tubing to
> come out of the chamber. The tubing is connected to a syringe pump
> outside the enclosure.
>
> Any suggestions with feedback on companies to checkout will be great.
>
> Thanks,
>
> -Prabhakar
>

Edrun Andrea Schnell

Deep penetration in tissue with two photon

In reply to this post by Holly L. AARON

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear list,

it is a known fact that a two photon laser will penetrate deeper into
tissue, and we have done several experiments to show this, but without
success. We have used spheroids labeled with several dyes, and imaged
with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two
photon laser and descanned detector and 3) two photon laser and
non-descanned detector. We have only found a difference of about 10-20 um
from exp. 1) to exp. 3), whereas it should be around 100 um.

So I'm wondering if anyone else have done this kind of experiment and have
any tips as to how to image this increase in penetration depth? Type of
sample and dye etc.?

Thanks a lot!

Regards,
Edrun A. Schnell

--

Edrun Andrea Schnell
Divisional engineer,
Dept. of Physics, NTNU
Hogskoleringen 5, 7491 Trondheim
Norway

Nuno Moreno

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Witch objective and mounting medium are you using? Is the laser passing
through an AOM?

Edrun Andrea Schnell wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list,
>
> it is a known fact that a two photon laser will penetrate deeper into
> tissue, and we have done several experiments to show this, but without
> success. We have used spheroids labeled with several dyes, and imaged
> with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two
> photon laser and descanned detector and 3) two photon laser and
> non-descanned detector. We have only found a difference of about 10-20 um
> from exp. 1) to exp. 3), whereas it should be around 100 um.
>
> So I'm wondering if anyone else have done this kind of experiment and have
> any tips as to how to image this increase in penetration depth? Type of
> sample and dye etc.?
>
> Thanks a lot!
>
> Regards,
> Edrun A. Schnell
>
> --
>
> Edrun Andrea Schnell
> Divisional engineer,
> Dept. of Physics, NTNU
> Hogskoleringen 5, 7491 Trondheim
> Norway
>

--
Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
http://uic.igc.gulbekian.pt
http://www.igc.gulbekian.pt
phone +351 214464606
fax +351 214407970

Edrun Andrea Schnell

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

We use the C-Apochromat 40x/1.2 W objective, which is supposed to be the
best for two photon, and yes, we do have an AOM. We don't use any mounting
medium since the spheroids are in solution (PBS, RPMI etc) in glass wells.

-Edrun

--

Edrun Andrea Schnell
Divisional engineer,
Dept. of Physics, NTNU
Hogskoleringen 5, 7491 Trondheim
Norway

On Fri, 7 Dec 2007, Nuno Moreno wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Witch objective and mounting medium are you using? Is the laser passing
> through an AOM?
>
> Edrun Andrea Schnell wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear list,
> >
> > it is a known fact that a two photon laser will penetrate deeper into
> > tissue, and we have done several experiments to show this, but without
> > success. We have used spheroids labeled with several dyes, and imaged
> > with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two
> > photon laser and descanned detector and 3) two photon laser and
> > non-descanned detector. We have only found a difference of about 10-20 um
> > from exp. 1) to exp. 3), whereas it should be around 100 um.
> >
> > So I'm wondering if anyone else have done this kind of experiment and have
> > any tips as to how to image this increase in penetration depth? Type of
> > sample and dye etc.?
> >
> > Thanks a lot!
> >
> > Regards,
> > Edrun A. Schnell
> >
> > --
> >
> > Edrun Andrea Schnell
> > Divisional engineer,
> > Dept. of Physics, NTNU
> > Hogskoleringen 5, 7491 Trondheim
> > Norway
> >
>
> --
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
> http://uic.igc.gulbekian.pt
> http://www.igc.gulbekian.pt
> phone +351 214464606
> fax +351 214407970
>

"José A. Feijó"

Re: Deep penetration in tissue with two photon

In reply to this post by Edrun Andrea Schnell

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

the problem I believe is not 2P physics, but the Zeiss 510. We've been
trying to keep a low profile about this, but it's certainly frustrating
that we can't really get much out of 510 under 2p, and it's painful that
we still get much better results with a side-by-side Bio-Rad 1024MP,
despite the 10yrs+ difference in terms of design and technology (do
mind, sharing the same laser and optical path, we just diverge form one
to the other with a simmple reflector!!). During the purchase process I
made a number of inquiries about a number of physical parameters
affecting the negative GVD performance of the system, namely the AOM and
the external detectors path, and NEVER got any satisfactory, even less
quantitative answer from anyone from Zeiss. The AOM sucks, it destroys
the power and the pulsewidth, one good reason for you to see such small
difference, penetration is pretty much dependent on both of these. We've
tried a simple hand operated ND filter, but unfortunately the fly-back
of the beam in the 510 turned out to be nasty for most samples. The
external PMT's optical design sucks^2, and does not help either.

In our hands, and after 2 years of fighting the beast and trying to have
some answers from Zeiss, we are changing our initial plan of downgrading
the Bio-Rad 1024 to confocal, and focus on the Zeiss for 2P to make use
of all the nice software features and (oh insane naivety!) eventually
the META detector, and now we find ourselves buying video amplifiers
from China to repair the Bio-Rad amplifiers and try to keep it running,
and eventually downgrade the Zeiss to bare confocal (eventually we will
have tow external for sale, in case anyone's interested...).

I voted against the merge Zeiss/BioRad, but was kind of hoping that
Zeiss people could eventually learn from their 2P design investment, and
their user database experience. That is surely not the case with the
510. In short I fail to meet anyone with a different experience than
ours, if there is someone out there on the list that actually managed to
bend a 510 to fulfill the kind of expectations we've got used to with
other machines, and have been reported in many papers out there, we
would be very interested in learning how. By now I have problems seeing
it coming from Zeiss, they are probably more concerned on developing the
forthcoming 610 or whatever (and then perhaps propose costly upgrades
"true that one didn't work, but this one will..."), but the sad reality
is that I feel that Zeiss has NOT come of age in matters of 2P. Not even
with the monopoly of the patent.

I would be very happy to retract myself to the list if I'm proven wrong
in this regard...

Jose

Edrun Andrea Schnell escreveu:

--

**********************************************************
Jose' A. Feijo', Prof.
----------------------------------------------------------
Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
PT-1749-016 Lisboa, PORTUGAL

tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48

and/ e

Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL

tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
__________________________________________________________
e.mail: [hidden email]
URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
**********************************************************

George McNamara

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Jose,

Thanks for the great post!

On City of Hope's multiphoton rig, with a Chameleon split between two
LSM510's (see Brian Armstrong's e-signature), I had no problem
imaging hundreds of um into freshly excised mouse brain stained with
Hoechst for 30 minutes (perfusion fixed - handy to flush the blood
out). 10x objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst
(do initial dilution from 10 mg/mL bottle into dH2O), then can dilute
into whatever the brain is in. 770 nm is also optimal for several
(not all!) Alexa dyes according to a JBO article by Mary Dickinson -
so Alexa Fluor 488 tomato lectin could be used to label blood vessels
immediately before sacrificing the mouse.

Edrun - This was with fairly low power, but don't be afraid to crank
up the power! Also, try more than one objective lens, and test
different wavelengths.

If working with fixed tissue: If you need to make a fixed specimen
transparent, there are several recipes for clearing tissue - see
Robert Zucker's papers using BABB for example. An interesting
mounting medium is 2,2'-thiodiethanol, published by Staudt et al in January.

best wishes,

George

At 06:28 AM 12/7/2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>the problem I believe is not 2P physics, but the Zeiss 510. We've
>been trying to keep a low profile about this, but it's certainly
>frustrating that we can't really get much out of 510 under 2p, and
>it's painful that we still get much better results with a
>side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
>of design and technology (do mind, sharing the same laser and
>optical path, we just diverge form one to the other with a simmple
>reflector!!). During the purchase process I made a number of
>inquiries about a number of physical parameters affecting the
>negative GVD performance of the system, namely the AOM and the
>external detectors path, and NEVER got any satisfactory, even less
>quantitative answer from anyone from Zeiss. The AOM sucks, it
>destroys the power and the pulsewidth, one good reason for you to
>see such small difference, penetration is pretty much dependent on
>both of these. We've tried a simple hand operated ND filter, but
>unfortunately the fly-back of the beam in the 510 turned out to be
>nasty for most samples. The external PMT's optical design sucks^2,
>and does not help either.
>
>In our hands, and after 2 years of fighting the beast and trying to
>have some answers from Zeiss, we are changing our initial plan of
>downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
>2P to make use of all the nice software features and (oh insane
>naivety!) eventually the META detector, and now we find ourselves
>buying video amplifiers from China to repair the Bio-Rad amplifiers
>and try to keep it running, and eventually downgrade the Zeiss to
>bare confocal (eventually we will have tow external for sale, in
>case anyone's interested...).
>
>I voted against the merge Zeiss/BioRad, but was kind of hoping that
>Zeiss people could eventually learn from their 2P design investment,
>and their user database experience. That is surely not the case with
>the 510. In short I fail to meet anyone with a different experience
>than ours, if there is someone out there on the list that actually
>managed to bend a 510 to fulfill the kind of expectations we've got
>used to with other machines, and have been reported in many papers
>out there, we would be very interested in learning how. By now I
>have problems seeing it coming from Zeiss, they are probably more
>concerned on developing the forthcoming 610 or whatever (and then
>perhaps propose costly upgrades "true that one didn't work, but this
>one will..."), but the sad reality is that I feel that Zeiss has NOT
>come of age in matters of 2P. Not even with the monopoly of the patent.
>
>I would be very happy to retract myself to the list if I'm proven
>wrong in this regard...
>
>Jose
>
>Edrun Andrea Schnell escreveu:
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Dear list,
>>
>>it is a known fact that a two photon laser will penetrate deeper into
>>tissue, and we have done several experiments to show this, but without
>>success. We have used spheroids labeled with several dyes, and imaged
>>with our Zeiss LSM510 with 1) visible laser and descanned detector, 2) two
>>photon laser and descanned detector and 3) two photon laser and
>>non-descanned detector. We have only found a difference of about 10-20 um
>>from exp. 1) to exp. 3), whereas it should be around 100 um.
>>
>>So I'm wondering if anyone else have done this kind of experiment and have
>>any tips as to how to image this increase in penetration depth? Type of
>>sample and dye etc.?
>>
>>Thanks a lot!
>>
>>Regards,
>>Edrun A. Schnell
>>
>>--
>>
>>Edrun Andrea Schnell
>>Divisional engineer,
>>Dept. of Physics, NTNU
>>Hogskoleringen 5, 7491 Trondheim
>>Norway
>>
>>
>
>--
>
>
>**********************************************************
>Jose' A. Feijo', Prof.
>----------------------------------------------------------
>Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>PT-1749-016 Lisboa, PORTUGAL
>
>tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>
>and/ e
>
>Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>
>tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>__________________________________________________________
>e.mail: [hidden email]
>URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>**********************************************************

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see
Analytical Imaging Core Facility)

"José A. Feijó"

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

thanks George, it's good to hear about good experiences (and definitely
you raise out some good points). But again 10x and 770nm may not be what
most people expect out of a 2P. And sure enough, Hoechst and DAPI are
"the" perfect 2P dyes!

George McNamara escreveu:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Jose,
>
> Thanks for the great post!
>
> On City of Hope's multiphoton rig, with a Chameleon split between two
> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
> hundreds of um into freshly excised mouse brain stained with Hoechst
> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
> dilution from 10 mg/mL bottle into dH2O), then can dilute into
> whatever the brain is in. 770 nm is also optimal for several (not
> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
> Alexa Fluor 488 tomato lectin could be used to label blood vessels
> immediately before sacrificing the mouse.
>
> Edrun - This was with fairly low power, but don't be afraid to crank
> up the power! Also, try more than one objective lens, and test
> different wavelengths.
>
> If working with fixed tissue: If you need to make a fixed specimen
> transparent, there are several recipes for clearing tissue - see
> Robert Zucker's papers using BABB for example. An interesting mounting
> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
>
> best wishes,
>
>
> George
>
>
>
> At 06:28 AM 12/7/2007, you wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> the problem I believe is not 2P physics, but the Zeiss 510. We've
>> been trying to keep a low profile about this, but it's certainly
>> frustrating that we can't really get much out of 510 under 2p, and
>> it's painful that we still get much better results with a
>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
>> of design and technology (do mind, sharing the same laser and optical
>> path, we just diverge form one to the other with a simmple
>> reflector!!). During the purchase process I made a number of
>> inquiries about a number of physical parameters affecting the
>> negative GVD performance of the system, namely the AOM and the
>> external detectors path, and NEVER got any satisfactory, even less
>> quantitative answer from anyone from Zeiss. The AOM sucks, it
>> destroys the power and the pulsewidth, one good reason for you to see
>> such small difference, penetration is pretty much dependent on both
>> of these. We've tried a simple hand operated ND filter, but
>> unfortunately the fly-back of the beam in the 510 turned out to be
>> nasty for most samples. The external PMT's optical design sucks^2,
>> and does not help either.
>>
>> In our hands, and after 2 years of fighting the beast and trying to
>> have some answers from Zeiss, we are changing our initial plan of
>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
>> 2P to make use of all the nice software features and (oh insane
>> naivety!) eventually the META detector, and now we find ourselves
>> buying video amplifiers from China to repair the Bio-Rad amplifiers
>> and try to keep it running, and eventually downgrade the Zeiss to
>> bare confocal (eventually we will have tow external for sale, in case
>> anyone's interested...).
>>
>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
>> Zeiss people could eventually learn from their 2P design investment,
>> and their user database experience. That is surely not the case with
>> the 510. In short I fail to meet anyone with a different experience
>> than ours, if there is someone out there on the list that actually
>> managed to bend a 510 to fulfill the kind of expectations we've got
>> used to with other machines, and have been reported in many papers
>> out there, we would be very interested in learning how. By now I
>> have problems seeing it coming from Zeiss, they are probably more
>> concerned on developing the forthcoming 610 or whatever (and then
>> perhaps propose costly upgrades "true that one didn't work, but this
>> one will..."), but the sad reality is that I feel that Zeiss has NOT
>> come of age in matters of 2P. Not even with the monopoly of the patent.
>>
>> I would be very happy to retract myself to the list if I'm proven
>> wrong in this regard...
>>
>> Jose
>>
>> Edrun Andrea Schnell escreveu:
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Dear list,
>>>
>>> it is a known fact that a two photon laser will penetrate deeper into
>>> tissue, and we have done several experiments to show this, but without
>>> success. We have used spheroids labeled with several dyes, and imaged
>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
>>> 2) two
>>> photon laser and descanned detector and 3) two photon laser and
>>> non-descanned detector. We have only found a difference of about
>>> 10-20 um
>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
>>>
>>> So I'm wondering if anyone else have done this kind of experiment
>>> and have
>>> any tips as to how to image this increase in penetration depth? Type of
>>> sample and dye etc.?
>>>
>>> Thanks a lot!
>>>
>>> Regards,
>>> Edrun A. Schnell
>>>
>>> --
>>>
>>> Edrun Andrea Schnell
>>> Divisional engineer,
>>> Dept. of Physics, NTNU
>>> Hogskoleringen 5, 7491 Trondheim
>>> Norway
>>>
>>>
>>
>> --
>>
>>
>> **********************************************************
>> Jose' A. Feijo', Prof.
>> ----------------------------------------------------------
>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>> PT-1749-016 Lisboa, PORTUGAL
>>
>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>
>> and/ e
>>
>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>
>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>> __________________________________________________________
>> e.mail: [hidden email]
>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>> **********************************************************
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> Analytical Imaging Core Facility)
>

Craig Brideau

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Our own lab has found that modifying confocal microscopes for 2-photon
is a tricky business. As mentioned before, an AOM will cause problems
with the laser. If there is any way to bypass the AOM in your system,
or simply remove it, this will help.
Another issue is optics and objectives. Most optics and objectives
are designed for the visible range rather than the NIR. From my own
experience certain medium-high-end Nikon and Olympus lenses tend to
have the best NIR performance, and most others are terrible; absorbing
90% or more of the light. A crude test is to use a laser power meter
to measure the power after your objective. Place the detector head
some distance away where the light is diverging but still all of it
hits the detector surface; the focal point can damage your detector.
Compare this to the power before the microscope to get your system
losses. Don't be afraid to ask your manufacturer for the NIR
transmission spectrum for your lenses.

Craig

On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> thanks George, it's good to hear about good experiences (and definitely
> you raise out some good points). But again 10x and 770nm may not be what
> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
> "the" perfect 2P dyes!
>
> George McNamara escreveu:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Hi Jose,
> >
> > Thanks for the great post!
> >
> > On City of Hope's multiphoton rig, with a Chameleon split between two
> > LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
> > hundreds of um into freshly excised mouse brain stained with Hoechst
> > for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
> > objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
> > dilution from 10 mg/mL bottle into dH2O), then can dilute into
> > whatever the brain is in. 770 nm is also optimal for several (not
> > all!) Alexa dyes according to a JBO article by Mary Dickinson - so
> > Alexa Fluor 488 tomato lectin could be used to label blood vessels
> > immediately before sacrificing the mouse.
> >
> > Edrun - This was with fairly low power, but don't be afraid to crank
> > up the power! Also, try more than one objective lens, and test
> > different wavelengths.
> >
> > If working with fixed tissue: If you need to make a fixed specimen
> > transparent, there are several recipes for clearing tissue - see
> > Robert Zucker's papers using BABB for example. An interesting mounting
> > medium is 2,2'-thiodiethanol, published by Staudt et al in January.
> >
> > best wishes,
> >
> >
> > George
> >
> >
> >
> > At 06:28 AM 12/7/2007, you wrote:
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> the problem I believe is not 2P physics, but the Zeiss 510. We've
> >> been trying to keep a low profile about this, but it's certainly
> >> frustrating that we can't really get much out of 510 under 2p, and
> >> it's painful that we still get much better results with a
> >> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
> >> of design and technology (do mind, sharing the same laser and optical
> >> path, we just diverge form one to the other with a simmple
> >> reflector!!). During the purchase process I made a number of
> >> inquiries about a number of physical parameters affecting the
> >> negative GVD performance of the system, namely the AOM and the
> >> external detectors path, and NEVER got any satisfactory, even less
> >> quantitative answer from anyone from Zeiss. The AOM sucks, it
> >> destroys the power and the pulsewidth, one good reason for you to see
> >> such small difference, penetration is pretty much dependent on both
> >> of these. We've tried a simple hand operated ND filter, but
> >> unfortunately the fly-back of the beam in the 510 turned out to be
> >> nasty for most samples. The external PMT's optical design sucks^2,
> >> and does not help either.
> >>
> >> In our hands, and after 2 years of fighting the beast and trying to
> >> have some answers from Zeiss, we are changing our initial plan of
> >> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
> >> 2P to make use of all the nice software features and (oh insane
> >> naivety!) eventually the META detector, and now we find ourselves
> >> buying video amplifiers from China to repair the Bio-Rad amplifiers
> >> and try to keep it running, and eventually downgrade the Zeiss to
> >> bare confocal (eventually we will have tow external for sale, in case
> >> anyone's interested...).
> >>
> >> I voted against the merge Zeiss/BioRad, but was kind of hoping that
> >> Zeiss people could eventually learn from their 2P design investment,
> >> and their user database experience. That is surely not the case with
> >> the 510. In short I fail to meet anyone with a different experience
> >> than ours, if there is someone out there on the list that actually
> >> managed to bend a 510 to fulfill the kind of expectations we've got
> >> used to with other machines, and have been reported in many papers
> >> out there, we would be very interested in learning how. By now I
> >> have problems seeing it coming from Zeiss, they are probably more
> >> concerned on developing the forthcoming 610 or whatever (and then
> >> perhaps propose costly upgrades "true that one didn't work, but this
> >> one will..."), but the sad reality is that I feel that Zeiss has NOT
> >> come of age in matters of 2P. Not even with the monopoly of the patent.
> >>
> >> I would be very happy to retract myself to the list if I'm proven
> >> wrong in this regard...
> >>
> >> Jose
> >>
> >> Edrun Andrea Schnell escreveu:
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> Dear list,
> >>>
> >>> it is a known fact that a two photon laser will penetrate deeper into
> >>> tissue, and we have done several experiments to show this, but without
> >>> success. We have used spheroids labeled with several dyes, and imaged
> >>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
> >>> 2) two
> >>> photon laser and descanned detector and 3) two photon laser and
> >>> non-descanned detector. We have only found a difference of about
> >>> 10-20 um
> >>> from exp. 1) to exp. 3), whereas it should be around 100 um.
> >>>
> >>> So I'm wondering if anyone else have done this kind of experiment
> >>> and have
> >>> any tips as to how to image this increase in penetration depth? Type of
> >>> sample and dye etc.?
> >>>
> >>> Thanks a lot!
> >>>
> >>> Regards,
> >>> Edrun A. Schnell
> >>>
> >>> --
> >>>
> >>> Edrun Andrea Schnell
> >>> Divisional engineer,
> >>> Dept. of Physics, NTNU
> >>> Hogskoleringen 5, 7491 Trondheim
> >>> Norway
> >>>
> >>>
> >>
> >> --
> >>
> >>
> >> **********************************************************
> >> Jose' A. Feijo', Prof.
> >> ----------------------------------------------------------
> >> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> >> PT-1749-016 Lisboa, PORTUGAL
> >>
> >> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
> >>
> >> and/ e
> >>
> >> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
> >>
> >> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> >> __________________________________________________________
> >> e.mail: [hidden email]
> >> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> >> **********************************************************
> >
> >
> >
> >
> >
> >
> > George McNamara, Ph.D.
> > University of Miami, Miller School of Medicine
> > Image Core
> > Miami, FL 33010
> > [hidden email]
> > [hidden email]
> > 305-243-8436 office
> > http://home.earthlink.net/~pubspectra/
> > http://home.earthlink.net/~geomcnamara/
> > http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> > Analytical Imaging Core Facility)
> >
>
> --
>
>
>
> **********************************************************
> Jose' A. Feijo', Prof.
> ----------------------------------------------------------
> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> PT-1749-016 Lisboa, PORTUGAL
>
> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>
> and/ e
>
> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>
> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> __________________________________________________________
> e.mail: [hidden email]
> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> **********************************************************
>

Nuno Moreno

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

In the Zeiss the problem of removing the AOM is that it has a mechanical
shutter that opens before the galvos start to scan. This means that you
will get a burned spot at the center of the FOV (I tried that one). You
can build some electronics to synchronize the slow motor/bar based
shutter with the AOM signals but I would believe it will be an pain...or
maybe not, I have to check that!

Talking about pre-chirping do you know someone using something similar
to maitai deepsee in a Coherent Mira laser?

Regards,
NM

Craig Brideau wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Our own lab has found that modifying confocal microscopes for 2-photon
> is a tricky business. As mentioned before, an AOM will cause problems
> with the laser. If there is any way to bypass the AOM in your system,
> or simply remove it, this will help.
> Another issue is optics and objectives. Most optics and objectives
> are designed for the visible range rather than the NIR. From my own
> experience certain medium-high-end Nikon and Olympus lenses tend to
> have the best NIR performance, and most others are terrible; absorbing
> 90% or more of the light. A crude test is to use a laser power meter
> to measure the power after your objective. Place the detector head
> some distance away where the light is diverging but still all of it
> hits the detector surface; the focal point can damage your detector.
> Compare this to the power before the microscope to get your system
> losses. Don't be afraid to ask your manufacturer for the NIR
> transmission spectrum for your lenses.
>
> Craig
>
>
> On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> thanks George, it's good to hear about good experiences (and definitely
>> you raise out some good points). But again 10x and 770nm may not be what
>> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
>> "the" perfect 2P dyes!
>>
>> George McNamara escreveu:
>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Hi Jose,
>>>
>>> Thanks for the great post!
>>>
>>> On City of Hope's multiphoton rig, with a Chameleon split between two
>>> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
>>> hundreds of um into freshly excised mouse brain stained with Hoechst
>>> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
>>> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
>>> dilution from 10 mg/mL bottle into dH2O), then can dilute into
>>> whatever the brain is in. 770 nm is also optimal for several (not
>>> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
>>> Alexa Fluor 488 tomato lectin could be used to label blood vessels
>>> immediately before sacrificing the mouse.
>>>
>>> Edrun - This was with fairly low power, but don't be afraid to crank
>>> up the power! Also, try more than one objective lens, and test
>>> different wavelengths.
>>>
>>> If working with fixed tissue: If you need to make a fixed specimen
>>> transparent, there are several recipes for clearing tissue - see
>>> Robert Zucker's papers using BABB for example. An interesting mounting
>>> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
>>>
>>> best wishes,
>>>
>>>
>>> George
>>>
>>>
>>>
>>> At 06:28 AM 12/7/2007, you wrote:
>>>> Search the CONFOCAL archive at
>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>> the problem I believe is not 2P physics, but the Zeiss 510. We've
>>>> been trying to keep a low profile about this, but it's certainly
>>>> frustrating that we can't really get much out of 510 under 2p, and
>>>> it's painful that we still get much better results with a
>>>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
>>>> of design and technology (do mind, sharing the same laser and optical
>>>> path, we just diverge form one to the other with a simmple
>>>> reflector!!). During the purchase process I made a number of
>>>> inquiries about a number of physical parameters affecting the
>>>> negative GVD performance of the system, namely the AOM and the
>>>> external detectors path, and NEVER got any satisfactory, even less
>>>> quantitative answer from anyone from Zeiss. The AOM sucks, it
>>>> destroys the power and the pulsewidth, one good reason for you to see
>>>> such small difference, penetration is pretty much dependent on both
>>>> of these. We've tried a simple hand operated ND filter, but
>>>> unfortunately the fly-back of the beam in the 510 turned out to be
>>>> nasty for most samples. The external PMT's optical design sucks^2,
>>>> and does not help either.
>>>>
>>>> In our hands, and after 2 years of fighting the beast and trying to
>>>> have some answers from Zeiss, we are changing our initial plan of
>>>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
>>>> 2P to make use of all the nice software features and (oh insane
>>>> naivety!) eventually the META detector, and now we find ourselves
>>>> buying video amplifiers from China to repair the Bio-Rad amplifiers
>>>> and try to keep it running, and eventually downgrade the Zeiss to
>>>> bare confocal (eventually we will have tow external for sale, in case
>>>> anyone's interested...).
>>>>
>>>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
>>>> Zeiss people could eventually learn from their 2P design investment,
>>>> and their user database experience. That is surely not the case with
>>>> the 510. In short I fail to meet anyone with a different experience
>>>> than ours, if there is someone out there on the list that actually
>>>> managed to bend a 510 to fulfill the kind of expectations we've got
>>>> used to with other machines, and have been reported in many papers
>>>> out there, we would be very interested in learning how. By now I
>>>> have problems seeing it coming from Zeiss, they are probably more
>>>> concerned on developing the forthcoming 610 or whatever (and then
>>>> perhaps propose costly upgrades "true that one didn't work, but this
>>>> one will..."), but the sad reality is that I feel that Zeiss has NOT
>>>> come of age in matters of 2P. Not even with the monopoly of the patent.
>>>>
>>>> I would be very happy to retract myself to the list if I'm proven
>>>> wrong in this regard...
>>>>
>>>> Jose
>>>>
>>>> Edrun Andrea Schnell escreveu:
>>>>> Search the CONFOCAL archive at
>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>> Dear list,
>>>>>
>>>>> it is a known fact that a two photon laser will penetrate deeper into
>>>>> tissue, and we have done several experiments to show this, but without
>>>>> success. We have used spheroids labeled with several dyes, and imaged
>>>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
>>>>> 2) two
>>>>> photon laser and descanned detector and 3) two photon laser and
>>>>> non-descanned detector. We have only found a difference of about
>>>>> 10-20 um
>>>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
>>>>>
>>>>> So I'm wondering if anyone else have done this kind of experiment
>>>>> and have
>>>>> any tips as to how to image this increase in penetration depth? Type of
>>>>> sample and dye etc.?
>>>>>
>>>>> Thanks a lot!
>>>>>
>>>>> Regards,
>>>>> Edrun A. Schnell
>>>>>
>>>>> --
>>>>>
>>>>> Edrun Andrea Schnell
>>>>> Divisional engineer,
>>>>> Dept. of Physics, NTNU
>>>>> Hogskoleringen 5, 7491 Trondheim
>>>>> Norway
>>>>>
>>>>>
>>>> --
>>>>
>>>>
>>>> **********************************************************
>>>> Jose' A. Feijo', Prof.
>>>> ----------------------------------------------------------
>>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>>>> PT-1749-016 Lisboa, PORTUGAL
>>>>
>>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>>>
>>>> and/ e
>>>>
>>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>>>
>>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>>>> __________________________________________________________
>>>> e.mail: [hidden email]
>>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>>>> **********************************************************
>>>
>>>
>>>
>>>
>>>
>>> George McNamara, Ph.D.
>>> University of Miami, Miller School of Medicine
>>> Image Core
>>> Miami, FL 33010
>>> [hidden email]
>>> [hidden email]
>>> 305-243-8436 office
>>> http://home.earthlink.net/~pubspectra/
>>> http://home.earthlink.net/~geomcnamara/
>>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>> Analytical Imaging Core Facility)
>>>
>> --
>>
>>
>>
>> **********************************************************
>> Jose' A. Feijo', Prof.
>> ----------------------------------------------------------
>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>> PT-1749-016 Lisboa, PORTUGAL
>>
>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>
>> and/ e
>>
>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>
>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>> __________________________________________________________
>> e.mail: [hidden email]
>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>> **********************************************************
>>
>

--
Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
http://uic.igc.gulbekian.pt
http://www.igc.gulbekian.pt
phone +351 214464606
fax +351 214407970

"José A. Feijó"

Re: Deep penetration in tissue with two photon

In reply to this post by Craig Brideau

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal we published some of our own evaluations of Nikon lenses in Protoplasma, 223:132, 2004
all the plan-fluo lenses are decisivly more transmissive the Plan-Apo, specially above 850 nm, in which the later are basically useless. We end up finding the better one being the S-Fluo, ironically developed for UV quantification purposes (don't know if it still exists in the Nikon catalogue). The situation may have changed though, all these were comercial lenses in 1999-2000. Meanwhile we found out that some Leica lenses make suprisingly good jobs as well in the Nikon/Bio-rad platform, so it's always usefull to spend some time with the available lenses and a real application, no matter how "strange" these optics marriages may sound in the beggining. We don't have experience with Olympus lenses, but they always had a reputation of high transmitance.

Craig Brideau escreveu:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Our own lab has found that modifying confocal microscopes for 2-photon
is a tricky business.  As mentioned before, an AOM will cause problems
with the laser.  If there is any way to bypass the AOM in your system,
or simply remove it, this will help.
Another issue is optics and objectives.  Most optics and objectives
are designed for the visible range rather than the NIR.  From my own
experience certain medium-high-end Nikon and Olympus lenses tend to
have the best NIR performance, and most others are terrible; absorbing
90% or more of the light.  A crude test is to use a laser power meter
to measure the power after your objective. Place the detector head
some distance away where the light is diverging but still all of it
hits the detector surface; the focal point can damage your detector.
Compare this to the power before the microscope to get your system
losses.  Don't be afraid to ask your manufacturer for the NIR
transmission spectrum for your lenses.

Craig


On Dec 7, 2007 5:06 AM, "José A. Feijó" [hidden email] wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

thanks George, it's good to hear about good experiences (and definitely
you raise out some good points). But again 10x and 770nm may not be what
most people expect out of a 2P. And sure enough, Hoechst and DAPI are
"the" perfect 2P dyes!

George McNamara escreveu:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Jose,

Thanks for the great post!

On City of Hope's multiphoton rig, with a Chameleon split between two
LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
hundreds of um into freshly excised mouse brain stained with Hoechst
for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
dilution from 10 mg/mL bottle into dH2O), then can dilute into
whatever the brain is in. 770 nm is also optimal for several (not
all!) Alexa dyes according to a JBO article by Mary Dickinson - so
Alexa Fluor 488 tomato lectin could be used to label blood vessels
immediately before sacrificing the mouse.

Edrun - This was with fairly low power, but don't be afraid to crank
up the power! Also, try more than one objective lens, and test
different wavelengths.

If working with fixed tissue: If you need to make a fixed specimen
transparent, there are several recipes for clearing tissue - see
Robert Zucker's papers using BABB for example. An interesting mounting
medium is 2,2'-thiodiethanol, published by Staudt et al in January.

best wishes,


George



At 06:28 AM 12/7/2007, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

the problem I believe is not 2P physics, but the Zeiss 510. We've
been trying to keep a low profile about this, but it's certainly
frustrating that we can't really get much out of 510 under 2p, and
it's painful that we still get much better results with a
side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
of design and technology (do mind, sharing the same laser and optical
path, we just diverge form one to the other with a simmple
reflector!!). During the purchase process I made a number of
inquiries about a number of physical parameters affecting the
negative GVD performance of the system, namely the AOM and the
external detectors path, and NEVER got any satisfactory, even less
quantitative answer from anyone from Zeiss. The AOM sucks, it
destroys the power and the pulsewidth, one good reason for you to see
such small difference, penetration is pretty much dependent on both
of these. We've tried a simple hand operated ND filter, but
unfortunately the fly-back of the beam in the 510 turned out to be
nasty for most samples. The external PMT's optical design sucks^2,
and does not help either.

In our hands, and after 2 years of fighting the beast and trying to
have some answers from Zeiss, we are changing our initial plan of
downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
2P to make use of all the nice software features and (oh insane
naivety!) eventually the META detector, and now we find ourselves
buying video amplifiers from China to repair the Bio-Rad amplifiers
and try to keep it running, and eventually downgrade the Zeiss to
bare confocal (eventually we will have tow external for sale, in case
anyone's interested...).

I voted against the merge Zeiss/BioRad, but was kind of hoping that
Zeiss people could eventually learn from their 2P design investment,
and their user database experience. That is surely not the case with
the 510. In short I fail to meet anyone with a different experience
than ours, if there is someone out there on the list that actually
managed to bend a 510 to fulfill the kind of expectations we've got
used to with other machines, and have been reported in many papers
out there, we would be very interested in learning how.  By now I
have problems seeing it coming from Zeiss, they are probably more
concerned on developing the forthcoming 610 or whatever (and then
perhaps propose costly upgrades "true that one didn't work, but this
one will..."), but the sad reality is that I feel that Zeiss has NOT
come of age in matters of 2P. Not even with the monopoly of the patent.

I would be very happy to retract myself to the list if I'm proven
wrong in this regard...

Jose

Edrun Andrea Schnell escreveu:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear list,

it is a known fact that a two photon laser will penetrate deeper into
tissue, and we have done several experiments to show this, but without
success. We have used spheroids labeled with several dyes, and imaged
with our Zeiss LSM510 with 1) visible laser and descanned detector,
2) two
photon laser and descanned detector and 3) two photon laser and
non-descanned detector. We have only found a difference of about
10-20 um
from exp. 1) to exp. 3), whereas it should be around 100 um.

So I'm wondering if anyone else have done this kind of experiment
and have
any tips as to how to image this increase in penetration depth? Type of
sample and dye etc.?

Thanks a lot!

Regards,
Edrun A. Schnell

--

Edrun Andrea Schnell
Divisional engineer,
Dept. of Physics, NTNU
Hogskoleringen 5, 7491 Trondheim
Norway

--


**********************************************************
Jose' A. Feijo', Prof.
----------------------------------------------------------
Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
PT-1749-016 Lisboa, PORTUGAL

tel. +351.21.750.00.47/00/24, fax  +351.21.750.00.48

and/ e

Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL

tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
__________________________________________________________
e.mail: [hidden email]
URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
**********************************************************





George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see
Analytical Imaging Core Facility)

--



**********************************************************
Jose' A. Feijo', Prof.
----------------------------------------------------------
Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
PT-1749-016 Lisboa, PORTUGAL

tel. +351.21.750.00.47/00/24, fax  +351.21.750.00.48

and/ e

Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL

tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
__________________________________________________________
e.mail: [hidden email]
URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
**********************************************************

-- 


**********************************************************
Jose' A. Feijo', Prof.                    
----------------------------------------------------------         
Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
PT-1749-016 Lisboa, PORTUGAL

tel. +351.21.750.00.47/00/24, fax  +351.21.750.00.48

and/ e

Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL

tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
__________________________________________________________
e.mail: [hidden email]                          
URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
**********************************************************

Craig Brideau

Re: Deep penetration in tissue with two photon

In reply to this post by Nuno Moreno

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Pre-chirp only makes a huge difference when the GVD induced by your
system is quite large. This is probably true when something like an
AOM is present. We built our own compressors with prisms and
optomechanics, but this is not an exercise for the faint-of-heart.
From what I've seen the DeepSee is probably the simplest GVD control
method from a user perspective and apparently can provide quite a high
level of GVD compensation.

Craig

On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> In the Zeiss the problem of removing the AOM is that it has a mechanical
> shutter that opens before the galvos start to scan. This means that you
> will get a burned spot at the center of the FOV (I tried that one). You
> can build some electronics to synchronize the slow motor/bar based
> shutter with the AOM signals but I would believe it will be an pain...or
> maybe not, I have to check that!
>
> Talking about pre-chirping do you know someone using something similar
> to maitai deepsee in a Coherent Mira laser?
>
> Regards,
> NM
>
>
> Craig Brideau wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Our own lab has found that modifying confocal microscopes for 2-photon
> > is a tricky business. As mentioned before, an AOM will cause problems
> > with the laser. If there is any way to bypass the AOM in your system,
> > or simply remove it, this will help.
> > Another issue is optics and objectives. Most optics and objectives
> > are designed for the visible range rather than the NIR. From my own
> > experience certain medium-high-end Nikon and Olympus lenses tend to
> > have the best NIR performance, and most others are terrible; absorbing
> > 90% or more of the light. A crude test is to use a laser power meter
> > to measure the power after your objective. Place the detector head
> > some distance away where the light is diverging but still all of it
> > hits the detector surface; the focal point can damage your detector.
> > Compare this to the power before the microscope to get your system
> > losses. Don't be afraid to ask your manufacturer for the NIR
> > transmission spectrum for your lenses.
> >
> > Craig
> >
> >
> > On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote:
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> thanks George, it's good to hear about good experiences (and definitely
> >> you raise out some good points). But again 10x and 770nm may not be what
> >> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
> >> "the" perfect 2P dyes!
> >>
> >> George McNamara escreveu:
> >>
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> Hi Jose,
> >>>
> >>> Thanks for the great post!
> >>>
> >>> On City of Hope's multiphoton rig, with a Chameleon split between two
> >>> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
> >>> hundreds of um into freshly excised mouse brain stained with Hoechst
> >>> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
> >>> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
> >>> dilution from 10 mg/mL bottle into dH2O), then can dilute into
> >>> whatever the brain is in. 770 nm is also optimal for several (not
> >>> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
> >>> Alexa Fluor 488 tomato lectin could be used to label blood vessels
> >>> immediately before sacrificing the mouse.
> >>>
> >>> Edrun - This was with fairly low power, but don't be afraid to crank
> >>> up the power! Also, try more than one objective lens, and test
> >>> different wavelengths.
> >>>
> >>> If working with fixed tissue: If you need to make a fixed specimen
> >>> transparent, there are several recipes for clearing tissue - see
> >>> Robert Zucker's papers using BABB for example. An interesting mounting
> >>> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
> >>>
> >>> best wishes,
> >>>
> >>>
> >>> George
> >>>
> >>>
> >>>
> >>> At 06:28 AM 12/7/2007, you wrote:
> >>>> Search the CONFOCAL archive at
> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>
> >>>> the problem I believe is not 2P physics, but the Zeiss 510. We've
> >>>> been trying to keep a low profile about this, but it's certainly
> >>>> frustrating that we can't really get much out of 510 under 2p, and
> >>>> it's painful that we still get much better results with a
> >>>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
> >>>> of design and technology (do mind, sharing the same laser and optical
> >>>> path, we just diverge form one to the other with a simmple
> >>>> reflector!!). During the purchase process I made a number of
> >>>> inquiries about a number of physical parameters affecting the
> >>>> negative GVD performance of the system, namely the AOM and the
> >>>> external detectors path, and NEVER got any satisfactory, even less
> >>>> quantitative answer from anyone from Zeiss. The AOM sucks, it
> >>>> destroys the power and the pulsewidth, one good reason for you to see
> >>>> such small difference, penetration is pretty much dependent on both
> >>>> of these. We've tried a simple hand operated ND filter, but
> >>>> unfortunately the fly-back of the beam in the 510 turned out to be
> >>>> nasty for most samples. The external PMT's optical design sucks^2,
> >>>> and does not help either.
> >>>>
> >>>> In our hands, and after 2 years of fighting the beast and trying to
> >>>> have some answers from Zeiss, we are changing our initial plan of
> >>>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
> >>>> 2P to make use of all the nice software features and (oh insane
> >>>> naivety!) eventually the META detector, and now we find ourselves
> >>>> buying video amplifiers from China to repair the Bio-Rad amplifiers
> >>>> and try to keep it running, and eventually downgrade the Zeiss to
> >>>> bare confocal (eventually we will have tow external for sale, in case
> >>>> anyone's interested...).
> >>>>
> >>>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
> >>>> Zeiss people could eventually learn from their 2P design investment,
> >>>> and their user database experience. That is surely not the case with
> >>>> the 510. In short I fail to meet anyone with a different experience
> >>>> than ours, if there is someone out there on the list that actually
> >>>> managed to bend a 510 to fulfill the kind of expectations we've got
> >>>> used to with other machines, and have been reported in many papers
> >>>> out there, we would be very interested in learning how. By now I
> >>>> have problems seeing it coming from Zeiss, they are probably more
> >>>> concerned on developing the forthcoming 610 or whatever (and then
> >>>> perhaps propose costly upgrades "true that one didn't work, but this
> >>>> one will..."), but the sad reality is that I feel that Zeiss has NOT
> >>>> come of age in matters of 2P. Not even with the monopoly of the patent.
> >>>>
> >>>> I would be very happy to retract myself to the list if I'm proven
> >>>> wrong in this regard...
> >>>>
> >>>> Jose
> >>>>
> >>>> Edrun Andrea Schnell escreveu:
> >>>>> Search the CONFOCAL archive at
> >>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>>
> >>>>> Dear list,
> >>>>>
> >>>>> it is a known fact that a two photon laser will penetrate deeper into
> >>>>> tissue, and we have done several experiments to show this, but without
> >>>>> success. We have used spheroids labeled with several dyes, and imaged
> >>>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
> >>>>> 2) two
> >>>>> photon laser and descanned detector and 3) two photon laser and
> >>>>> non-descanned detector. We have only found a difference of about
> >>>>> 10-20 um
> >>>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
> >>>>>
> >>>>> So I'm wondering if anyone else have done this kind of experiment
> >>>>> and have
> >>>>> any tips as to how to image this increase in penetration depth? Type of
> >>>>> sample and dye etc.?
> >>>>>
> >>>>> Thanks a lot!
> >>>>>
> >>>>> Regards,
> >>>>> Edrun A. Schnell
> >>>>>
> >>>>> --
> >>>>>
> >>>>> Edrun Andrea Schnell
> >>>>> Divisional engineer,
> >>>>> Dept. of Physics, NTNU
> >>>>> Hogskoleringen 5, 7491 Trondheim
> >>>>> Norway
> >>>>>
> >>>>>
> >>>> --
> >>>>
> >>>>
> >>>> **********************************************************
> >>>> Jose' A. Feijo', Prof.
> >>>> ----------------------------------------------------------
> >>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> >>>> PT-1749-016 Lisboa, PORTUGAL
> >>>>
> >>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
> >>>>
> >>>> and/ e
> >>>>
> >>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
> >>>>
> >>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> >>>> __________________________________________________________
> >>>> e.mail: [hidden email]
> >>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> >>>> **********************************************************
> >>>
> >>>
> >>>
> >>>
> >>>
> >>> George McNamara, Ph.D.
> >>> University of Miami, Miller School of Medicine
> >>> Image Core
> >>> Miami, FL 33010
> >>> [hidden email]
> >>> [hidden email]
> >>> 305-243-8436 office
> >>> http://home.earthlink.net/~pubspectra/
> >>> http://home.earthlink.net/~geomcnamara/
> >>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> >>> Analytical Imaging Core Facility)
> >>>
> >> --
> >>
> >>
> >>
> >> **********************************************************
> >> Jose' A. Feijo', Prof.
> >> ----------------------------------------------------------
> >> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> >> PT-1749-016 Lisboa, PORTUGAL
> >>
> >> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
> >>
> >> and/ e
> >>
> >> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
> >>
> >> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> >> __________________________________________________________
> >> e.mail: [hidden email]
> >> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> >> **********************************************************
> >>
> >
>
> --
>
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
> http://uic.igc.gulbekian.pt
> http://www.igc.gulbekian.pt
> phone +351 214464606
> fax +351 214407970
>

Maria Smedh

Re: Deep penetration in tissue with two photon

In reply to this post by Edrun Andrea Schnell

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Edrun,

How deep are you trying to go? At least our old C-Apochromat 40x/1.2 W
objectives only have a working distance of ~0.3 mm (which have been debated
here on the list whether it is counted from the top lens element or from the
cover glass)...

/Maria

~~~~~~~~~~~~~~~~~~

Maria Smedh, Ph.D.

Centre for Cellular Imaging
The Sahlgrenska Academy, Göteborg University
Visiting address: Medicinareg. 7A, 413 90 Göteborg
Postal address: Box 435, 40530 Göteborg
Delivery address: Medicinaregatan 1G, 413 90 Göteborg, Sweden

Phone: + 46 (0)31 786 3704
Mobile: + 46 (0)70 298 3424
E-mail: [hidden email]

Webpage: http://www.cf.gu.se/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Edrun Andrea Schnell
Sent: den 7 december 2007 12:20
To: [hidden email]
Subject: Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

We use the C-Apochromat 40x/1.2 W objective, which is supposed to be the
best for two photon, and yes, we do have an AOM. We don't use any mounting
medium since the spheroids are in solution (PBS, RPMI etc) in glass wells.

-Edrun

--

Edrun Andrea Schnell
Divisional engineer,
Dept. of Physics, NTNU
Hogskoleringen 5, 7491 Trondheim
Norway

On Fri, 7 Dec 2007, Nuno Moreno wrote:

two
> > photon laser and descanned detector and 3) two photon laser and
> > non-descanned detector. We have only found a difference of about 10-20
um
> > from exp. 1) to exp. 3), whereas it should be around 100 um.
> >
> > So I'm wondering if anyone else have done this kind of experiment and
have

> > any tips as to how to image this increase in penetration depth? Type of
> > sample and dye etc.?
> >
> > Thanks a lot!
> >
> > Regards,
> > Edrun A. Schnell
> >
> > --
> >
> > Edrun Andrea Schnell
> > Divisional engineer,
> > Dept. of Physics, NTNU
> > Hogskoleringen 5, 7491 Trondheim
> > Norway
> >
>
> --
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
> http://uic.igc.gulbekian.pt
> http://www.igc.gulbekian.pt
> phone +351 214464606
> fax +351 214407970
>

Armstrong, Brian

Re: Deep penetration in tissue with two photon

In reply to this post by Craig Brideau

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

After I heard a presentation about the Deep See I spent some time researching pre-chirp and talked with some Coherent people (we have 2 Chameleon's) about this as well as some people I respect in 2P imaging. In short, the consensus is that it really does not gain you very much. Coherent does not have a plan to pre-chirp (as conveyed to me). Moreover, the physics suggests that you need to vary the inter-prism distance with lambda changes. I asked the people at LaVision how they accomplish this but was not very satisfied with their answers (they sell an MMM scope that has a pre-chirped beam). I wonder how useful the gain in signal is from pre-chirping when you have a laser that puts out 3W of power. Yes, I realize that it is not quite the same, but certainly increased power can compensate somewhat.
I guess you could circumvent the AOM in the Zeiss system. The AOM adjusts the percent power to the scope, to do this would necessitate adding a pockels cell.
Happy Holidays,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
http://www.cityofhope.org/SharedResources/LightMicroscopy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, December 07, 2007 7:21 AM
To: [hidden email]
Subject: Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Pre-chirp only makes a huge difference when the GVD induced by your
system is quite large. This is probably true when something like an
AOM is present. We built our own compressors with prisms and
optomechanics, but this is not an exercise for the faint-of-heart.
From what I've seen the DeepSee is probably the simplest GVD control
method from a user perspective and apparently can provide quite a high
level of GVD compensation.

Craig

On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote:

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Nuno Moreno

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I think that coherent is losing on this. Having more power means fried
cells. For sure that having more photons arriving at once will increase
the probability of 2P absorption! Increasing power will increase this
probability but will also increase the energy delivered to your
sample...and this is high-school physics, P=E/T. The same energy
arriving in a shorter period will increase the peak power and you need
it since 2P cross sections are large orders of magnitude below 1P

Regards,
NM

Armstrong, Brian wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> After I heard a presentation about the Deep See I spent some time researching pre-chirp and talked with some Coherent people (we have 2 Chameleon's) about this as well as some people I respect in 2P imaging. In short, the consensus is that it really does not gain you very much. Coherent does not have a plan to pre-chirp (as conveyed to me). Moreover, the physics suggests that you need to vary the inter-prism distance with lambda changes. I asked the people at LaVision how they accomplish this but was not very satisfied with their answers (they sell an MMM scope that has a pre-chirped beam). I wonder how useful the gain in signal is from pre-chirping when you have a laser that puts out 3W of power. Yes, I realize that it is not quite the same, but certainly increased power can compensate somewhat.
> I guess you could circumvent the AOM in the Zeiss system. The AOM adjusts the percent power to the scope, to do this would necessitate adding a pockels cell.
> Happy Holidays,
>
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-359-8111 x62872
> http://www.cityofhope.org/SharedResources/LightMicroscopy
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, December 07, 2007 7:21 AM
> To: [hidden email]
> Subject: Re: Deep penetration in tissue with two photon
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Pre-chirp only makes a huge difference when the GVD induced by your
> system is quite large. This is probably true when something like an
> AOM is present. We built our own compressors with prisms and
> optomechanics, but this is not an exercise for the faint-of-heart.
> From what I've seen the DeepSee is probably the simplest GVD control
> method from a user perspective and apparently can provide quite a high
> level of GVD compensation.
>
> Craig
>
>
> On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi,
>>
>> In the Zeiss the problem of removing the AOM is that it has a mechanical
>> shutter that opens before the galvos start to scan. This means that you
>> will get a burned spot at the center of the FOV (I tried that one). You
>> can build some electronics to synchronize the slow motor/bar based
>> shutter with the AOM signals but I would believe it will be an pain...or
>> maybe not, I have to check that!
>>
>> Talking about pre-chirping do you know someone using something similar
>> to maitai deepsee in a Coherent Mira laser?
>>
>> Regards,
>> NM
>>
>>
>> Craig Brideau wrote:
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Our own lab has found that modifying confocal microscopes for 2-photon
>>> is a tricky business. As mentioned before, an AOM will cause problems
>>> with the laser. If there is any way to bypass the AOM in your system,
>>> or simply remove it, this will help.
>>> Another issue is optics and objectives. Most optics and objectives
>>> are designed for the visible range rather than the NIR. From my own
>>> experience certain medium-high-end Nikon and Olympus lenses tend to
>>> have the best NIR performance, and most others are terrible; absorbing
>>> 90% or more of the light. A crude test is to use a laser power meter
>>> to measure the power after your objective. Place the detector head
>>> some distance away where the light is diverging but still all of it
>>> hits the detector surface; the focal point can damage your detector.
>>> Compare this to the power before the microscope to get your system
>>> losses. Don't be afraid to ask your manufacturer for the NIR
>>> transmission spectrum for your lenses.
>>>
>>> Craig
>>>
>>>
>>> On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote:
>>>> Search the CONFOCAL archive at
>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>> thanks George, it's good to hear about good experiences (and definitely
>>>> you raise out some good points). But again 10x and 770nm may not be what
>>>> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
>>>> "the" perfect 2P dyes!
>>>>
>>>> George McNamara escreveu:
>>>>
>>>>> Search the CONFOCAL archive at
>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>> Hi Jose,
>>>>>
>>>>> Thanks for the great post!
>>>>>
>>>>> On City of Hope's multiphoton rig, with a Chameleon split between two
>>>>> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
>>>>> hundreds of um into freshly excised mouse brain stained with Hoechst
>>>>> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
>>>>> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
>>>>> dilution from 10 mg/mL bottle into dH2O), then can dilute into
>>>>> whatever the brain is in. 770 nm is also optimal for several (not
>>>>> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
>>>>> Alexa Fluor 488 tomato lectin could be used to label blood vessels
>>>>> immediately before sacrificing the mouse.
>>>>>
>>>>> Edrun - This was with fairly low power, but don't be afraid to crank
>>>>> up the power! Also, try more than one objective lens, and test
>>>>> different wavelengths.
>>>>>
>>>>> If working with fixed tissue: If you need to make a fixed specimen
>>>>> transparent, there are several recipes for clearing tissue - see
>>>>> Robert Zucker's papers using BABB for example. An interesting mounting
>>>>> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
>>>>>
>>>>> best wishes,
>>>>>
>>>>>
>>>>> George
>>>>>
>>>>>
>>>>>
>>>>> At 06:28 AM 12/7/2007, you wrote:
>>>>>> Search the CONFOCAL archive at
>>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>>
>>>>>> the problem I believe is not 2P physics, but the Zeiss 510. We've
>>>>>> been trying to keep a low profile about this, but it's certainly
>>>>>> frustrating that we can't really get much out of 510 under 2p, and
>>>>>> it's painful that we still get much better results with a
>>>>>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
>>>>>> of design and technology (do mind, sharing the same laser and optical
>>>>>> path, we just diverge form one to the other with a simmple
>>>>>> reflector!!). During the purchase process I made a number of
>>>>>> inquiries about a number of physical parameters affecting the
>>>>>> negative GVD performance of the system, namely the AOM and the
>>>>>> external detectors path, and NEVER got any satisfactory, even less
>>>>>> quantitative answer from anyone from Zeiss. The AOM sucks, it
>>>>>> destroys the power and the pulsewidth, one good reason for you to see
>>>>>> such small difference, penetration is pretty much dependent on both
>>>>>> of these. We've tried a simple hand operated ND filter, but
>>>>>> unfortunately the fly-back of the beam in the 510 turned out to be
>>>>>> nasty for most samples. The external PMT's optical design sucks^2,
>>>>>> and does not help either.
>>>>>>
>>>>>> In our hands, and after 2 years of fighting the beast and trying to
>>>>>> have some answers from Zeiss, we are changing our initial plan of
>>>>>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
>>>>>> 2P to make use of all the nice software features and (oh insane
>>>>>> naivety!) eventually the META detector, and now we find ourselves
>>>>>> buying video amplifiers from China to repair the Bio-Rad amplifiers
>>>>>> and try to keep it running, and eventually downgrade the Zeiss to
>>>>>> bare confocal (eventually we will have tow external for sale, in case
>>>>>> anyone's interested...).
>>>>>>
>>>>>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
>>>>>> Zeiss people could eventually learn from their 2P design investment,
>>>>>> and their user database experience. That is surely not the case with
>>>>>> the 510. In short I fail to meet anyone with a different experience
>>>>>> than ours, if there is someone out there on the list that actually
>>>>>> managed to bend a 510 to fulfill the kind of expectations we've got
>>>>>> used to with other machines, and have been reported in many papers
>>>>>> out there, we would be very interested in learning how. By now I
>>>>>> have problems seeing it coming from Zeiss, they are probably more
>>>>>> concerned on developing the forthcoming 610 or whatever (and then
>>>>>> perhaps propose costly upgrades "true that one didn't work, but this
>>>>>> one will..."), but the sad reality is that I feel that Zeiss has NOT
>>>>>> come of age in matters of 2P. Not even with the monopoly of the patent.
>>>>>>
>>>>>> I would be very happy to retract myself to the list if I'm proven
>>>>>> wrong in this regard...
>>>>>>
>>>>>> Jose
>>>>>>
>>>>>> Edrun Andrea Schnell escreveu:
>>>>>>> Search the CONFOCAL archive at
>>>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>>>
>>>>>>> Dear list,
>>>>>>>
>>>>>>> it is a known fact that a two photon laser will penetrate deeper into
>>>>>>> tissue, and we have done several experiments to show this, but without
>>>>>>> success. We have used spheroids labeled with several dyes, and imaged
>>>>>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
>>>>>>> 2) two
>>>>>>> photon laser and descanned detector and 3) two photon laser and
>>>>>>> non-descanned detector. We have only found a difference of about
>>>>>>> 10-20 um
>>>>>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
>>>>>>>
>>>>>>> So I'm wondering if anyone else have done this kind of experiment
>>>>>>> and have
>>>>>>> any tips as to how to image this increase in penetration depth? Type of
>>>>>>> sample and dye etc.?
>>>>>>>
>>>>>>> Thanks a lot!
>>>>>>>
>>>>>>> Regards,
>>>>>>> Edrun A. Schnell
>>>>>>>
>>>>>>> --
>>>>>>>
>>>>>>> Edrun Andrea Schnell
>>>>>>> Divisional engineer,
>>>>>>> Dept. of Physics, NTNU
>>>>>>> Hogskoleringen 5, 7491 Trondheim
>>>>>>> Norway
>>>>>>>
>>>>>>>
>>>>>> --
>>>>>>
>>>>>>
>>>>>> **********************************************************
>>>>>> Jose' A. Feijo', Prof.
>>>>>> ----------------------------------------------------------
>>>>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>>>>>> PT-1749-016 Lisboa, PORTUGAL
>>>>>>
>>>>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>>>>>
>>>>>> and/ e
>>>>>>
>>>>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>>>>>
>>>>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>>>>>> __________________________________________________________
>>>>>> e.mail: [hidden email]
>>>>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>>>>>> **********************************************************
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> George McNamara, Ph.D.
>>>>> University of Miami, Miller School of Medicine
>>>>> Image Core
>>>>> Miami, FL 33010
>>>>> [hidden email]
>>>>> [hidden email]
>>>>> 305-243-8436 office
>>>>> http://home.earthlink.net/~pubspectra/
>>>>> http://home.earthlink.net/~geomcnamara/
>>>>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
>>>>> Analytical Imaging Core Facility)
>>>>>
>>>> --
>>>>
>>>>
>>>>
>>>> **********************************************************
>>>> Jose' A. Feijo', Prof.
>>>> ----------------------------------------------------------
>>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
>>>> PT-1749-016 Lisboa, PORTUGAL
>>>>
>>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
>>>>
>>>> and/ e
>>>>
>>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
>>>>
>>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
>>>> __________________________________________________________
>>>> e.mail: [hidden email]
>>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
>>>> **********************************************************
>>>>
>> --
>>
>> Nuno Moreno
>> Cell Imaging Unit
>> Instituto Gulbenkian de Ciência
>> http://uic.igc.gulbekian.pt
>> http://www.igc.gulbekian.pt
>> phone +351 214464606
>> fax +351 214407970
>>
>
>
> ---------------------------------------------------------------------
>
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for he individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not w

ish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
>
> ---------------------------------------------------------------------
>

--
Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
http://uic.igc.gulbekian.pt
http://www.igc.gulbekian.pt
phone +351 214464606
fax +351 214407970

Craig Brideau

Re: Deep penetration in tissue with two photon

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

There's actually an interesting balance on this. Coherent's lasers
have what I would call 'medium' pulsewidths of about 140 fs. The
bandwidth corresponding to this pulsewidth is 'relatively' narrow so
the pulse does not disperse 'much' when it passes through a typical
microscope system. Spectra Physics' lasers have a broader bandwidth,
which means that their pulses can be compressed 'shorter' (<90fs or
so) but they are also more sensitive to dispersion. Basically with an
SP laser and a dispersive system you really need GVD compensation, but
you will also really benefit from it as your pulses can actually be
quite short at the sample since the initial pulse contains the large
bandwidth necessary to support it. On the other hand, if your system
is only moderately dispersive the Coherent laser sidesteps the issue
by having a smaller bandwidth from the start. Each system has its
purpose: for something medium-low dispersive I would go Coherent, but
for something with buckets of dispersion I might actually consider a
DeepSee since you can compress for optimal performance in addition to
just compensating for the dispersion of your system.

Craig

On Dec 7, 2007 11:15 AM, Nuno Moreno <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I think that coherent is losing on this. Having more power means fried
> cells. For sure that having more photons arriving at once will increase
> the probability of 2P absorption! Increasing power will increase this
> probability but will also increase the energy delivered to your
> sample...and this is high-school physics, P=E/T. The same energy
> arriving in a shorter period will increase the peak power and you need
> it since 2P cross sections are large orders of magnitude below 1P
>
> Regards,
> NM
>
>
> Armstrong, Brian wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > After I heard a presentation about the Deep See I spent some time researching pre-chirp and talked with some Coherent people (we have 2 Chameleon's) about this as well as some people I respect in 2P imaging. In short, the consensus is that it really does not gain you very much. Coherent does not have a plan to pre-chirp (as conveyed to me). Moreover, the physics suggests that you need to vary the inter-prism distance with lambda changes. I asked the people at LaVision how they accomplish this but was not very satisfied with their answers (they sell an MMM scope that has a pre-chirped beam). I wonder how useful the gain in signal is from pre-chirping when you have a laser that puts out 3W of power. Yes, I realize that it is not quite the same, but certainly increased power can compensate somewhat.
> > I guess you could circumvent the AOM in the Zeiss system. The AOM adjusts the percent power to the scope, to do this would necessitate adding a pockels cell.
> > Happy Holidays,
> >
> > Brian D Armstrong PhD
> > Light Microscopy Core Manager
> > Beckman Research Institute
> > City of Hope
> > 1450 E Duarte Rd
> > Duarte, CA 91010
> > 626-359-8111 x62872
> > http://www.cityofhope.org/SharedResources/LightMicroscopy
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> > Sent: Friday, December 07, 2007 7:21 AM
> > To: [hidden email]
> > Subject: Re: Deep penetration in tissue with two photon
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Pre-chirp only makes a huge difference when the GVD induced by your
> > system is quite large. This is probably true when something like an
> > AOM is present. We built our own compressors with prisms and
> > optomechanics, but this is not an exercise for the faint-of-heart.
> > From what I've seen the DeepSee is probably the simplest GVD control
> > method from a user perspective and apparently can provide quite a high
> > level of GVD compensation.
> >
> > Craig
> >
> >
> > On Dec 7, 2007 7:32 AM, Nuno Moreno <[hidden email]> wrote:
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Hi,
> >>
> >> In the Zeiss the problem of removing the AOM is that it has a mechanical
> >> shutter that opens before the galvos start to scan. This means that you
> >> will get a burned spot at the center of the FOV (I tried that one). You
> >> can build some electronics to synchronize the slow motor/bar based
> >> shutter with the AOM signals but I would believe it will be an pain...or
> >> maybe not, I have to check that!
> >>
> >> Talking about pre-chirping do you know someone using something similar
> >> to maitai deepsee in a Coherent Mira laser?
> >>
> >> Regards,
> >> NM
> >>
> >>
> >> Craig Brideau wrote:
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> Our own lab has found that modifying confocal microscopes for 2-photon
> >>> is a tricky business. As mentioned before, an AOM will cause problems
> >>> with the laser. If there is any way to bypass the AOM in your system,
> >>> or simply remove it, this will help.
> >>> Another issue is optics and objectives. Most optics and objectives
> >>> are designed for the visible range rather than the NIR. From my own
> >>> experience certain medium-high-end Nikon and Olympus lenses tend to
> >>> have the best NIR performance, and most others are terrible; absorbing
> >>> 90% or more of the light. A crude test is to use a laser power meter
> >>> to measure the power after your objective. Place the detector head
> >>> some distance away where the light is diverging but still all of it
> >>> hits the detector surface; the focal point can damage your detector.
> >>> Compare this to the power before the microscope to get your system
> >>> losses. Don't be afraid to ask your manufacturer for the NIR
> >>> transmission spectrum for your lenses.
> >>>
> >>> Craig
> >>>
> >>>
> >>> On Dec 7, 2007 5:06 AM, "José A. Feijó" <[hidden email]> wrote:
> >>>> Search the CONFOCAL archive at
> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>
> >>>> thanks George, it's good to hear about good experiences (and definitely
> >>>> you raise out some good points). But again 10x and 770nm may not be what
> >>>> most people expect out of a 2P. And sure enough, Hoechst and DAPI are
> >>>> "the" perfect 2P dyes!
> >>>>
> >>>> George McNamara escreveu:
> >>>>
> >>>>> Search the CONFOCAL archive at
> >>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>>
> >>>>> Hi Jose,
> >>>>>
> >>>>> Thanks for the great post!
> >>>>>
> >>>>> On City of Hope's multiphoton rig, with a Chameleon split between two
> >>>>> LSM510's (see Brian Armstrong's e-signature), I had no problem imaging
> >>>>> hundreds of um into freshly excised mouse brain stained with Hoechst
> >>>>> for 30 minutes (perfusion fixed - handy to flush the blood out). 10x
> >>>>> objective lens. Try 770 nm excitation, 10 ug/mL Hoeschst (do initial
> >>>>> dilution from 10 mg/mL bottle into dH2O), then can dilute into
> >>>>> whatever the brain is in. 770 nm is also optimal for several (not
> >>>>> all!) Alexa dyes according to a JBO article by Mary Dickinson - so
> >>>>> Alexa Fluor 488 tomato lectin could be used to label blood vessels
> >>>>> immediately before sacrificing the mouse.
> >>>>>
> >>>>> Edrun - This was with fairly low power, but don't be afraid to crank
> >>>>> up the power! Also, try more than one objective lens, and test
> >>>>> different wavelengths.
> >>>>>
> >>>>> If working with fixed tissue: If you need to make a fixed specimen
> >>>>> transparent, there are several recipes for clearing tissue - see
> >>>>> Robert Zucker's papers using BABB for example. An interesting mounting
> >>>>> medium is 2,2'-thiodiethanol, published by Staudt et al in January.
> >>>>>
> >>>>> best wishes,
> >>>>>
> >>>>>
> >>>>> George
> >>>>>
> >>>>>
> >>>>>
> >>>>> At 06:28 AM 12/7/2007, you wrote:
> >>>>>> Search the CONFOCAL archive at
> >>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>>>
> >>>>>> the problem I believe is not 2P physics, but the Zeiss 510. We've
> >>>>>> been trying to keep a low profile about this, but it's certainly
> >>>>>> frustrating that we can't really get much out of 510 under 2p, and
> >>>>>> it's painful that we still get much better results with a
> >>>>>> side-by-side Bio-Rad 1024MP, despite the 10yrs+ difference in terms
> >>>>>> of design and technology (do mind, sharing the same laser and optical
> >>>>>> path, we just diverge form one to the other with a simmple
> >>>>>> reflector!!). During the purchase process I made a number of
> >>>>>> inquiries about a number of physical parameters affecting the
> >>>>>> negative GVD performance of the system, namely the AOM and the
> >>>>>> external detectors path, and NEVER got any satisfactory, even less
> >>>>>> quantitative answer from anyone from Zeiss. The AOM sucks, it
> >>>>>> destroys the power and the pulsewidth, one good reason for you to see
> >>>>>> such small difference, penetration is pretty much dependent on both
> >>>>>> of these. We've tried a simple hand operated ND filter, but
> >>>>>> unfortunately the fly-back of the beam in the 510 turned out to be
> >>>>>> nasty for most samples. The external PMT's optical design sucks^2,
> >>>>>> and does not help either.
> >>>>>>
> >>>>>> In our hands, and after 2 years of fighting the beast and trying to
> >>>>>> have some answers from Zeiss, we are changing our initial plan of
> >>>>>> downgrading the Bio-Rad 1024 to confocal, and focus on the Zeiss for
> >>>>>> 2P to make use of all the nice software features and (oh insane
> >>>>>> naivety!) eventually the META detector, and now we find ourselves
> >>>>>> buying video amplifiers from China to repair the Bio-Rad amplifiers
> >>>>>> and try to keep it running, and eventually downgrade the Zeiss to
> >>>>>> bare confocal (eventually we will have tow external for sale, in case
> >>>>>> anyone's interested...).
> >>>>>>
> >>>>>> I voted against the merge Zeiss/BioRad, but was kind of hoping that
> >>>>>> Zeiss people could eventually learn from their 2P design investment,
> >>>>>> and their user database experience. That is surely not the case with
> >>>>>> the 510. In short I fail to meet anyone with a different experience
> >>>>>> than ours, if there is someone out there on the list that actually
> >>>>>> managed to bend a 510 to fulfill the kind of expectations we've got
> >>>>>> used to with other machines, and have been reported in many papers
> >>>>>> out there, we would be very interested in learning how. By now I
> >>>>>> have problems seeing it coming from Zeiss, they are probably more
> >>>>>> concerned on developing the forthcoming 610 or whatever (and then
> >>>>>> perhaps propose costly upgrades "true that one didn't work, but this
> >>>>>> one will..."), but the sad reality is that I feel that Zeiss has NOT
> >>>>>> come of age in matters of 2P. Not even with the monopoly of the patent.
> >>>>>>
> >>>>>> I would be very happy to retract myself to the list if I'm proven
> >>>>>> wrong in this regard...
> >>>>>>
> >>>>>> Jose
> >>>>>>
> >>>>>> Edrun Andrea Schnell escreveu:
> >>>>>>> Search the CONFOCAL archive at
> >>>>>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>>>>>
> >>>>>>> Dear list,
> >>>>>>>
> >>>>>>> it is a known fact that a two photon laser will penetrate deeper into
> >>>>>>> tissue, and we have done several experiments to show this, but without
> >>>>>>> success. We have used spheroids labeled with several dyes, and imaged
> >>>>>>> with our Zeiss LSM510 with 1) visible laser and descanned detector,
> >>>>>>> 2) two
> >>>>>>> photon laser and descanned detector and 3) two photon laser and
> >>>>>>> non-descanned detector. We have only found a difference of about
> >>>>>>> 10-20 um
> >>>>>>> from exp. 1) to exp. 3), whereas it should be around 100 um.
> >>>>>>>
> >>>>>>> So I'm wondering if anyone else have done this kind of experiment
> >>>>>>> and have
> >>>>>>> any tips as to how to image this increase in penetration depth? Type of
> >>>>>>> sample and dye etc.?
> >>>>>>>
> >>>>>>> Thanks a lot!
> >>>>>>>
> >>>>>>> Regards,
> >>>>>>> Edrun A. Schnell
> >>>>>>>
> >>>>>>> --
> >>>>>>>
> >>>>>>> Edrun Andrea Schnell
> >>>>>>> Divisional engineer,
> >>>>>>> Dept. of Physics, NTNU
> >>>>>>> Hogskoleringen 5, 7491 Trondheim
> >>>>>>> Norway
> >>>>>>>
> >>>>>>>
> >>>>>> --
> >>>>>>
> >>>>>>
> >>>>>> **********************************************************
> >>>>>> Jose' A. Feijo', Prof.
> >>>>>> ----------------------------------------------------------
> >>>>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> >>>>>> PT-1749-016 Lisboa, PORTUGAL
> >>>>>>
> >>>>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
> >>>>>>
> >>>>>> and/ e
> >>>>>>
> >>>>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
> >>>>>>
> >>>>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> >>>>>> __________________________________________________________
> >>>>>> e.mail: [hidden email]
> >>>>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> >>>>>> **********************************************************
> >>>>>
> >>>>>
> >>>>>
> >>>>>
> >>>>> George McNamara, Ph.D.
> >>>>> University of Miami, Miller School of Medicine
> >>>>> Image Core
> >>>>> Miami, FL 33010
> >>>>> [hidden email]
> >>>>> [hidden email]
> >>>>> 305-243-8436 office
> >>>>> http://home.earthlink.net/~pubspectra/
> >>>>> http://home.earthlink.net/~geomcnamara/
> >>>>> http://www.sylvester.org/health_pro/shared_resources/index.asp (see
> >>>>> Analytical Imaging Core Facility)
> >>>>>
> >>>> --
> >>>>
> >>>>
> >>>>
> >>>> **********************************************************
> >>>> Jose' A. Feijo', Prof.
> >>>> ----------------------------------------------------------
> >>>> Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa
> >>>> PT-1749-016 Lisboa, PORTUGAL
> >>>>
> >>>> tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48
> >>>>
> >>>> and/ e
> >>>>
> >>>> Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL
> >>>>
> >>>> tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70
> >>>> __________________________________________________________
> >>>> e.mail: [hidden email]
> >>>> URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38
> >>>> **********************************************************
> >>>>
> >> --
> >>
> >> Nuno Moreno
> >> Cell Imaging Unit
> >> Instituto Gulbenkian de Ciência
> >> http://uic.igc.gulbekian.pt
> >> http://www.igc.gulbekian.pt
> >> phone +351 214464606
> >> fax +351 214407970
> >>
> >
> >
> > ---------------------------------------------------------------------
> >
> > SECURITY/CONFIDENTIALITY WARNING:
> > This message and any attachments are intended solely for he individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not w
> ish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
> >
> > ---------------------------------------------------------------------
> >
>
> --
>
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
> http://uic.igc.gulbekian.pt
> http://www.igc.gulbekian.pt
> phone +351 214464606
> fax +351 214407970
>

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