Acquiring a multiphoton system-some questions

[Jan Grawe]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

we are in the process  of acquiring a multiphoton
system intended mainly for intravital imaging,
and I have a couple of questions regarding performance:

1. If I understand it correctly (correct me
otherwise!),  intensity modulation using an AOM
is a major source of chirp. At least one
manufacturer offers an alternative without AOM,
which is said to improve imaging depth. With the
advent of pre-chirping, is this a relevant issue?
We would of course like the utility and bleaching
capabilities afforded by an AOM, but perhaps not
if it significantly reduces the imaging depth.

2. Connected to the above, to those of you that
have experience with pre-chirping. How much could
we expect this to improve our imaging depth? Is
the improvement greater if we choose a laser with
shorter pulses ~80fs as compared to 140fs?

2. Since no manufacturer will give any hard
numbers with regard to the depth reach of their
systems, we would like to be able to test this
for ourselves. Do you know of a test sample that
can be made several hundred um thick, that is or
mimics some type of tissue, that has specific
fluorescent structures at various depths, and
that preferably is stable at least over some
weeks. Are there commercially available MP test samples?
A tall order maybe, but I´m hoping..

4. What components of the system do you feel are
the most critical for the overall performance. Is
it the laser pulse shape, NDD performance, obcectives, other??


All responses are appreciated!

Best regards,

Jan Grawé




Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys

[George McNamara]

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

Hi Jan,

If you get a Coherent Chameleon II with 3.3 Watts (average, my recollection is that a 100 fs pulse repeated at 100 MHz is 1e6 higher peak, so 200 fs pulses would be 5e5 peak) power - or its Spectra-Physics equivalent - the depth limit is the burning smell of the surface of the tissue (see also Kleinfeld and Tsien's laser ablation article, pubmed 12848930 - I'm curious whether they breathed the ablated molecules).

Overall performance: NDD location (and PMT quality) and objective lenses.

You may want to talk with Martin Kohler in Per-Olaf Berggren's group at the Karolinska about intravital MP imaging. See http://ki.se/ki/jsp/polopoly.jsp?l=sv&d=2061 (full disclosure: PO also works at my location http://www.diabetesresearch.org/AbouttheDRI/DRIFaculty/PerOlofBerggren.htm from his lack of tan, the photo was probably taken at the beginning of one of his visits).


Most people I've asked about pre-chirp have told me it is not worth the bother. I've gone a about 1 mm into a freshly removed mouse brain (Hoechst staining for a few minutes, did perfuse to get rid of the blood) - I think the limit was either how far the (Zeiss 10x lens focused or how much time we were willing to spend acquiring one field of view). A group in the UK - see

Girkin JM, McConnell G.
 
Advances in laser sources for confocal and multiphoton microscopy.
Microsc Res Tech. 2005 May;67(1):8-14. Review.
PMID: 16025485


for example, is one of the few who are into short pulses (how many diseases have they cured with it?). The other use of pre-chirp is with LaVision Biotec's TriMScope, but it is for high speed calcium imaging not deep tissue imaging.



At 04:28 PM 2/24/2008, you wrote:

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

Dear all,

we are in the process  of acquiring a multiphoton system intended mainly for intravital imaging, and I have a couple of questions regarding performance:

1. If I understand it correctly (correct me otherwise!),  intensity modulation using an AOM is a major source of chirp. At least one manufacturer offers an alternative without AOM, which is said to improve imaging depth. With the advent of pre-chirping, is this a relevant issue? We would of course like the utility and bleaching capabilities afforded by an AOM, but perhaps not if it significantly reduces the imaging depth.

2. Connected to the above, to those of you that have experience with pre-chirping. How much could we expect this to improve our imaging depth? Is the improvement greater if we choose a laser with shorter pulses ~80fs as compared to 140fs?

2. Since no manufacturer will give any hard numbers with regard to the depth reach of their systems, we would like to be able to test this for ourselves. Do you know of a test sample that can be made several hundred um thick, that is or mimics some type of tissue, that has specific fluorescent structures at various depths, and that preferably is stable at least over some weeks. Are there commercially available MP test samples?
A tall order maybe, but I´m hoping..

4. What components of the system do you feel are the most critical for the overall performance. Is it the laser pulse shape, NDD performance, obcectives, other??


All responses are appreciated!

Best regards,

Jan Grawé




Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys

 

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/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility)

[Craig Brideau]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you are trying to approach 1mm in depth, pre-chirp will matter.  If you are only going 300-500 um approx then you might be able to just crank up your power instead.  If you have an AOM I'd recommend pre-chirping.  The shorter your pulse is out of the laser the more it will benefit from pre-chirping.  Right now there are a few options that I've personally investigated (this is not a complete list by any means!):

Coherent makes the Chameleon, which is about 140fs out of the laser.  This laser is the least broadened by AOMs, glass in prisms/lenses in the microscope, etc. because it starts out with a fairly broad transform-limited pulse.  If you don't have much technical confidence, then this is the simplest way to go, but may not give optimal results depending on how deep you really need to go.

Spectra Physics makes the Mai Tai which typically has 70-80fs out of the laser.  It will be vulnerable to chirp, but at the same time if it is used with pre-chirping (prism compressor or the like) it is capable of good depth performance.  Systems using lasers like this have gone to 700um or so (check papers).

For a more 'far out' solution, I saw a nifty gadget from Coherent at this past Photonics West show.  It's a 10 or 20fs laser with a pre-chirping module add on/built-in.  You could probably go pretty deep with this thing, but it would require some adjustment of the chirp parameters.  The pulse is so short that it will be very sensitive to chirping.  The one drawback is that it is not tunable, although at 10fs the bandwidth (spectral content) of the laser is already quite broad so it will most likely stimulate most dyes.  If you want to be able to 'tune off' a dye though to check something else it wouldn't be possible with this laser.

Craig

On Sun, Feb 24, 2008 at 2:28 PM, Jan Grawé <[hidden email]> wrote:

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

Dear all,

we are in the process  of acquiring a multiphoton
system intended mainly for intravital imaging,
and I have a couple of questions regarding performance:

1. If I understand it correctly (correct me
otherwise!),  intensity modulation using an AOM
is a major source of chirp. At least one
manufacturer offers an alternative without AOM,
which is said to improve imaging depth. With the
advent of pre-chirping, is this a relevant issue?
We would of course like the utility and bleaching
capabilities afforded by an AOM, but perhaps not
if it significantly reduces the imaging depth.

2. Connected to the above, to those of you that
have experience with pre-chirping. How much could
we expect this to improve our imaging depth? Is
the improvement greater if we choose a laser with
shorter pulses ~80fs as compared to 140fs?

2. Since no manufacturer will give any hard
numbers with regard to the depth reach of their
systems, we would like to be able to test this
for ourselves. Do you know of a test sample that
can be made several hundred um thick, that is or
mimics some type of tissue, that has specific
fluorescent structures at various depths, and
that preferably is stable at least over some
weeks. Are there commercially available MP test samples?
A tall order maybe, but I´m hoping..

4. What components of the system do you feel are
the most critical for the overall performance. Is
it the laser pulse shape, NDD performance, obcectives, other??


All responses are appreciated!

Best regards,

Jan Grawé




Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys

[Adrian Smith-6]

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

On 25/02/2008, at 10:23 AM, George McNamara wrote:

The other use of pre-chirp is with LaVision Biotec's TriMScope, but it is for high speed calcium imaging not deep tissue imaging.

LaVision Biotec systems can also be equipped with PMTs and used in single beam mode where they are very suitable for deep tissue imaging (we have one for that purpose).

Performance in multi-beam mode with CCD is reported to be equivalent to single-beam mode at depths up to 200um. PMT-mode is said to have advantages beyond 300um. See - 10.1529/biophysj.106.102459

(there is also <http://www.blackwell-synergy.com/doi/abs/10.1111/j.1600-065X.2008.00582.x> but I've not got a copy yet).

Regards,

Adrian Smith

Centenary Institute, Sydney, Australia

[Armstrong, Brian]

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

Hi Jan, 1. I am having Zeiss and Coherent come out together to hopefully gain some insight into this question. Certainly the AOM will cause GVD, how much is the real question. 2. I agree with George that experts suggest that pre-chirping isn't really worth the bother. One question that I have never received a satisfactory answer to is how you adjust the pre-chirp every time you change lamda, the interprism distance would need to be altered to account for the differing chirp from the differing lambda. Both Spectra (MaiTai Deep See) and Coherent, offer pre-chirp options for their lasers.
3. Carolina makes prepared slides that are very thick. I have the pig embryo. http://www.carolina.com
4. That is an excellent question. It troubles me. I hear that Prairie Tech http://www.prairie-technologies.com/ "hand picks" their Hamamatsu PMTs. I think they are identical to the ones Zeiss used in their LSM510 NDD systems. I don't know if this would account for the difference, but the Prairie system is much more sensitive than the 510 NDD system. Zeiss will have new and improved (custom made?) PMTs on their LSM 710s. We will have to get the specs as they slowly leak out from Zeiss.
   

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 Jan Grawé
Sent: Sunday, February 24, 2008 1:28 PM
To: [hidden email]
Subject: Acquiring a multiphoton system-some questions

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

Dear all,

we are in the process  of acquiring a multiphoton
system intended mainly for intravital imaging,
and I have a couple of questions regarding performance:

1. If I understand it correctly (correct me
otherwise!),  intensity modulation using an AOM
is a major source of chirp. At least one
manufacturer offers an alternative without AOM,
which is said to improve imaging depth. With the
advent of pre-chirping, is this a relevant issue?
We would of course like the utility and bleaching
capabilities afforded by an AOM, but perhaps not
if it significantly reduces the imaging depth.

2. Connected to the above, to those of you that
have experience with pre-chirping. How much could
we expect this to improve our imaging depth? Is
the improvement greater if we choose a laser with
shorter pulses ~80fs as compared to 140fs?

2. Since no manufacturer will give any hard
numbers with regard to the depth reach of their
systems, we would like to be able to test this
for ourselves. Do you know of a test sample that
can be made several hundred um thick, that is or
mimics some type of tissue, that has specific
fluorescent structures at various depths, and
that preferably is stable at least over some
weeks. Are there commercially available MP test samples?
A tall order maybe, but I´m hoping..

4. What components of the system do you feel are
the most critical for the overall performance. Is
it the laser pulse shape, NDD performance, obcectives, other??


All responses are appreciated!

Best regards,

Jan Grawé




Jan Grawé
Cell Analysis Core Facility
Rudbecklaboratoriet/C5
Dag hammarskjölds väg 20
SE-75185 Uppsala
SWEDEN

Phone: +(0)18-4714657
Cell:   +(0)70-2577874
[hidden email]
www.rudbeck.uu.se/cellanalys


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