Re: 2-Photon System

[Neeraj Gohad-2]

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

Dear List,

Our lab is planning to acquire a 2-Photon microscope system in next couple of months for studying marine invertebrates. We are considering systems from different vendors such Thor labs, Sutter Instruments, Prairie Technologies, LaVision BioTec etc. We have imaged our samples on some of the systems. We have budget constraints and want to make the best possible choice so before we decide on any one vendor I am interested in hearing from folks who have one to these systems and use it for 2P imaging. How do you rate these systems on criteria like ease of use, ability to modify and expand system capabilities, efficacy of the software, servicing issues and vendor response to such issues, overall system integration etc.

Thanks in advance,

Best Regards,

Neeraj.


Neeraj V. Gohad, Ph.D.
Research Assistant Professor
Department of Biological Sciences
132 Long Hall
Clemson University
Clemson,SC-29634
Phone: [phone]
Fax: [phone]

[George McNamara]

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

Hi Neeraj,

I am very happy with our Coherent Chameleon Ultra II laser on our Leica
MP/SP5 multiphoton/confocal microscope (upright fixed stage Leica
scope). The microscope (in confocal mode) was the main instrument used
for the recently published "living window" model paper:

    High-resolution, noninvasive longitudinal live imaging of immune
    responses. </pubmed/21768391> Abdulreda MH, Faleo G, Molano RD,
    Lopez-Cabezas M, Molina J, Tan Y, Echeverria OA, Zahr-Akrawi E,
    Rodriguez-Diaz R, Edlund PK, Leibiger I, Bayer AL, Perez V, Ricordi
    C, *Caicedo* A, Pileggi A, Berggren PO. Proc Natl Acad Sci U S A.
    2011 Aug 2;108(31):12863-8. Epub 2011 Jul 18. PMID: 21768391.
    Intravital imaging emerged as an indispensible tool in biological
    research, and a variety of imaging techniques have been developed to
    noninvasively monitor tissues in vivo. However, most of the current
    techniques lack the resolution to study events at the single-cell
    level. Although intravital multiphoton microscopy has addressed this
    limitation, the need for repeated noninvasive access to the same
    tissue in longitudinal in vivo studies remains largely unmet. We now
    report on a previously unexplored approach to study immune responses
    after transplantation of pancreatic islets into the anterior chamber
    of the mouse eye. This approach enabled (i) longitudinal,
    noninvasive imaging of transplanted tissues in vivo; (ii) in vivo
    cytolabeling to assess cellular phenotype and viability in situ;
    (iii) local intervention by topical application or intraocular
    injection; and (iv) real-time tracking of infiltrating immune cells
    in the target tissue.


The MP laser service contract is a "must have". Coherent is 10% of
purchase price per year (after year 1 warranty). A demo Chameleon
visited in August with 4.8 W peak power - you could probably drive 4
microscopes with this (get a big enough table or plan on buying a new
one). Our Chameleon was refurbished in 4/2010 under service contract:
less than one day to replace the old one, 2.2 W peak power with the
factory refurbished to new (at that time) specification unit with 3.8 W.
The listserv has a long history of fans and bashers of Coherent and
Spectra-Physics, the two main players in the MP laser world. Ask your
salespeople for a COMPLETE LIST of customers who own the same laser that
you want to buy and are in your territory and call or email several for
comments.
The Scanner service contract is a "must have". Leica is 5% of purchase
price for first chunk of money ($400K?), then 4% there after. Our is
pretty fully loaded.

I am a long time fan of Sutter - I have been using and supporting their
filter wheels since 1991. For that matter, I've been using their pipette
pullers since 1981.
The LaVision TriMscope is an interesting approach to go fast. Our Leica
SP5 has both high res and resonant scanner - the latter is 8,000 lines
per second.

If you are not planning to use the scope 40 hours every week, consider
making it a core facility available to other labs on campus. There
probably are not a lot of MP scopes on campus. A useful accessory may be
the LSMtech objective inverter
http://www.lsmtech.com/product_objective_inverter.html

Leica and my core have an agreement that makes possible them doing
demo's on our scope. You can discuss this with your Leica confocal sales
rep. You might as well get a quote for instrument plus each year's
service contract to figure out if it is even in the range of your budget.

***

I am working to decommission our Zeiss LSM510 scope. Depending on your
Zeiss rep (and "the factory") and/or local expertise, you might be able
to buy our system, have Zeiss upgrade it to MP, at a price even less
than a new scanner. The LSM510 is still running and the LSM
software/electronics are a bit cranky - presumably the "factory" could
make it behave better. Our LSM510 is not the only old confocal around -
if you can inventory your campus' confocals you might be able to find
one that would make sense to move to your lab and upgrade to MP.


***

If you are doing high resolution imaging (1.4 NA), a couple of comments:
1. get the right vibration isolation table. You will not get 214 nm XY
resolution on a $5000 table in most buildings. Think 300 mm thick
breadboard, big fat legs.
2. If you need high resolution images, primarily, and especially if you
want to "colocalize" molecules, you need a nanoscope, not a microscope.
For examples, see:

    Nearest neighbor analysis of dopamine D1 receptors and Na(+) -K(+)
    -ATPases in dendritic spines dissected by *STED* microscopy.
    </pubmed/21809413>

    *Blom* H, Rönnlund D, Scott L, Spicarova Z, Rantanen V, Widengren J,
    Aperia A, Brismar H.

    Microsc Res Tech. 2011 Aug 1. doi: 10.1002/jemt.21046. [Epub ahead
    of print]

    PMID:
        21809413

    and

    Probing protein heterogeneity in the plasma membrane using PALM and
    pair correlation analysis. </pubmed/21926998>

    Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL,
    *Lippincott-Schwartz J*.

    Nat Methods. 2011 Sep 18. doi: 10.1038/nmeth.1704. [Epub ahead of print]

    PMID:
        21926998



Best wishes,


George



On 10/4/2011 2:36 PM, Neeraj Gohad wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear List,
>
> Our lab is planning to acquire a 2-Photon microscope system in next couple of months for studying marine invertebrates. We are considering systems from different vendors such Thor labs, Sutter Instruments, Prairie Technologies, LaVision BioTec etc. We have imaged our samples on some of the systems. We have budget constraints and want to make the best possible choice so before we decide on any one vendor I am interested in hearing from folks who have one to these systems and use it for 2P imaging. How do you rate these systems on criteria like ease of use, ability to modify and expand system capabilities, efficacy of the software, servicing issues and vendor response to such issues, overall system integration etc.
>
> Thanks in advance,
>
> Best Regards,
>
> Neeraj.
>
>
> Neeraj V. Gohad, Ph.D.
> Research Assistant Professor
> Department of Biological Sciences
> 132 Long Hall
> Clemson University
> Clemson,SC-29634
> Phone: [phone]
> Fax: [phone]
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami