WEB SEMINAR: A Deeper Understanding of In Vivo Immune Cell Function Through the Use of Multiphoton Intravital Microscopy

[Jeff Stuckey]

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Bruker Fluorescence Microscopy Webinar Series

Friday, June 13, 2014
10AM PT / 6PM GMT Register here:   https://www2.gotomeeting.com/register/318231746

Bruker Fluorescence Microscopy is pleased to have as a guest speaker this month Thorsten Mempel, MD, PhD, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital.

Dr. Mempel will present data showing how Multiphoton Intravital Microscopy (MP-IVM) is proving to be a revolutionary tool in the study of immune cell function, allowing observation of immune cell functions in lymphoid as well as non-lymphoid tissues, such as sites of infection or tumors. In addition to qualitative observation of immune cell behavior, MP-IVM lends itself to quantitative measurements of cell migration, cell-cell interactions and the activity of signaling pathways.

Data will focus on two areas of study:

1) normal T-lymphocyte function, specifically the interaction of cytotoxic T-cells with antigen-presenting cells at effector sites

2) exploitation of immune cells by pathogens, specifically HIV-1, to establish and maintain infection.

The benefits of in vivo methods compared to in vitro methods will be discussed, as well as general considerations for the use of MP-IVM in immune system research.




Jeff Stuckey PhD
Product Marketing Manager

Bruker Nano Surfaces Division
Fluorescence Microscopy Business Unit      [hidden email]<mailto:[hidden email]>
3030 Laura Lane, Suite 140                              www.bruker.com/nano<https://remote.bruker-nano.com/itweb/www.bruker.com/nano>
Middleton, WI 53562-0677
Phone: [phone] x162
________________________________
We are now Bruker!
Same great products.  Same great technology.
Same great people.  New great name.

[Quoc Thang Nguyen-2]

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Dear List members,

We would like to choose a dry objective lens (x20 to x40) for multiphoton microscopy with a working distance of at least 0.6 mm, 1-2 mm being ideal. Good NA and decent transmittance in NIR (we’ll excite mostly between 800 nm and 940 nm) are required.

Do you have an opinion on the following lenses (in the order of WD)?

ZEISS Plan-Achromat 20X
                NA 0.75
                WD 0.6 mm (a tad short)
NIKON CFI Super (5) Fluor 20
                NA 0.75
                WD: 1.00 mm
NIKON N20X-PF - 20X Plan Fluorite Imaging Objective
                NA: 0.50
                WD: 2.1 mm
NIKON CFI Plan Fluor 20X
                NA 0.50
                WD: 3.00 mm
OLYMPUS LUCPLFNL 40x
                NA: 0.6
                WD 2.7-4 mm

Thanks for the information.

Best regards to all,

Quoc

[Lu Yan]

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For NIR application, I had expression that Mitutoyo usually beats others. I
could be wrong though.

-----------------------------------------------------
Lu Yan
Nanostructured Fibers and Nonlinear Optics Laboratory
Electrical and Computer Engineering
Boston University
8 St. Mary St., Boston, MA, 02215
[phone]
[hidden email]
-----------------------------------------------------

On Fri, Oct 24, 2014 at 5:41 PM, Quoc Thang Nguyen <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List members,
>
> We would like to choose a dry objective lens (x20 to x40) for multiphoton
> microscopy with a working distance of at least 0.6 mm, 1-2 mm being ideal.
> Good NA and decent transmittance in NIR (we’ll excite mostly between 800 nm
> and 940 nm) are required.
>
> Do you have an opinion on the following lenses (in the order of WD)?
>
> ZEISS Plan-Achromat 20X
>                 NA 0.75
>                 WD 0.6 mm (a tad short)
> NIKON CFI Super (5) Fluor 20
>                 NA 0.75
>                 WD: 1.00 mm
> NIKON N20X-PF - 20X Plan Fluorite Imaging Objective
>                 NA: 0.50
>                 WD: 2.1 mm
> NIKON CFI Plan Fluor 20X
>                 NA 0.50
>                 WD: 3.00 mm
> OLYMPUS LUCPLFNL 40x
>                 NA: 0.6
>                 WD 2.7-4 mm
>
> Thanks for the information.
>
> Best regards to all,
>
> Quoc

[Craig Brideau]

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I've used a Mitutoyo 50x NIR HR. It does not handle coverslips well though;
it's meant for direct imaging of the sample with no intervening glass. It
has an NA of 0.65 and excellent transmission from the visible to around
1800nm I believe. It cost around $8k when I bought mine a few years back.

Craig


On Fri, Oct 24, 2014 at 5:27 PM, Yan, Lu <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> For NIR application, I had expression that Mitutoyo usually beats others. I
> could be wrong though.
>
> -----------------------------------------------------
> Lu Yan
> Nanostructured Fibers and Nonlinear Optics Laboratory
> Electrical and Computer Engineering
> Boston University
> 8 St. Mary St., Boston, MA, 02215
> [phone]
> [hidden email]
> -----------------------------------------------------
>
> On Fri, Oct 24, 2014 at 5:41 PM, Quoc Thang Nguyen <
> [hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Dear List members,
> >
> > We would like to choose a dry objective lens (x20 to x40) for multiphoton
> > microscopy with a working distance of at least 0.6 mm, 1-2 mm being
> ideal.
> > Good NA and decent transmittance in NIR (we’ll excite mostly between 800
> nm
> > and 940 nm) are required.
> >
> > Do you have an opinion on the following lenses (in the order of WD)?
> >
> > ZEISS Plan-Achromat 20X
> >                 NA 0.75
> >                 WD 0.6 mm (a tad short)
> > NIKON CFI Super (5) Fluor 20
> >                 NA 0.75
> >                 WD: 1.00 mm
> > NIKON N20X-PF - 20X Plan Fluorite Imaging Objective
> >                 NA: 0.50
> >                 WD: 2.1 mm
> > NIKON CFI Plan Fluor 20X
> >                 NA 0.50
> >                 WD: 3.00 mm
> > OLYMPUS LUCPLFNL 40x
> >                 NA: 0.6
> >                 WD 2.7-4 mm
> >
> > Thanks for the information.
> >
> > Best regards to all,
> >
> > Quoc
>

[Michael Giacomelli]

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If you want long working distance and only moderate NA, I would think about
20 or even 10x rather than 40x.  Zeiss sells a few inexpensive plan-fluor
10x objectives at 0.45 to 0.5 NA with long working distances and good NIR
transmission.   They will be a factor of several cheaper than many
dedicated 2 photon objectives, although obviously at lower NA.

Mike



On Fri, Oct 24, 2014 at 5:41 PM, Quoc Thang Nguyen <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List members,
>
> We would like to choose a dry objective lens (x20 to x40) for multiphoton
> microscopy with a working distance of at least 0.6 mm, 1-2 mm being ideal.
> Good NA and decent transmittance in NIR (we’ll excite mostly between 800 nm
> and 940 nm) are required.
>
> Do you have an opinion on the following lenses (in the order of WD)?
>
> ZEISS Plan-Achromat 20X
>                 NA 0.75
>                 WD 0.6 mm (a tad short)
> NIKON CFI Super (5) Fluor 20
>                 NA 0.75
>                 WD: 1.00 mm
> NIKON N20X-PF - 20X Plan Fluorite Imaging Objective
>                 NA: 0.50
>                 WD: 2.1 mm
> NIKON CFI Plan Fluor 20X
>                 NA 0.50
>                 WD: 3.00 mm
> OLYMPUS LUCPLFNL 40x
>                 NA: 0.6
>                 WD 2.7-4 mm
>
> Thanks for the information.
>
> Best regards to all,
>
> Quoc

[George McNamara]

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

For more light collection, consider adding "TED" ...

Compact non-contact total emission detection for in vivo *multiphoton*
excitation microscopy. <http://www.ncbi.nlm.nih.gov/pubmed/24251437>

Combs CA, Smirnov A, Glancy B, Karamzadeh NS, Gandjbakhche AH, Redford
G, Kilborn K, Knutson JR, *Balaban* RS.

J Microsc. 2014 Feb;253(2):83-92. doi: 10.1111/jmi.12099. Epub 2013 Nov 19.

PMID:
    24251437


We describe a compact, non-contact design for a total emission detection
(c-TED) system for intra-vital *multiphoton* imaging. To conform to a
standard upright two-photon microscope design, this system uses a
parabolic mirror surrounding a standard microscope objective in concert
with an optical path that does not interfere with normal microscope
operation. The non-contact design of this device allows for maximal
light collection without disrupting the physiology of the specimen being
examined. Tests were conducted on exposed tissues in live animals to
examine the emission collection enhancement of the c-TED device compared
to heavily optimized objective-based emission collection. The best light
collection enhancement was seen from murine fat (5×-2× gains as a
function of depth), whereas murine skeletal muscle and rat kidney showed
gains of over two and just under twofold near the surface, respectively.
Gains decreased with imaging depth (particularly in the kidney).
Zebrafish imaging on a reflective substrate showed close to a twofold
gain throughout the entire volume of an intact embryo (approximately 150
?m deep). Direct measurement of bleaching rates confirmed that the lower
laser powers, enabled by greater light collection efficiency, yielded
reduced photobleaching in vivo. The potential benefits of increased
light collection in terms of speed of imaging and reduced photo-damage,
as well as the applicability of this device to other *multiphoton*
imaging methods is discussed.

Published 2013. This article is a U.S. Government work and is in the
public domain in the USA.


Optimizing *multiphoton* fluorescence microscopy light collection from
living tissue by noncontact total emission detection (epiTED).
<http://www.ncbi.nlm.nih.gov/pubmed/21118209>

Combs CA, Smirnov A, Chess D, McGavern DB, Schroeder JL, Riley J, Kang
SS, Lugar-Hammer M, Gandjbakhche A, Knutson JR, *Balaban* RS.

J Microsc. 2011 Feb;241(2):153-61. doi:
10.1111/j.1365-2818.2010.03411.x. Epub 2010 Jun 21. Erratum in: J
Microsc. 2011 Aug;243(2):220.

PMID:
    21118209


A benefit of *multiphoton* fluorescence microscopy is the inherent
optical sectioning that occurs during excitation at the
diffraction-limited spot. The scanned collection of fluorescence
emission is incoherent; that is, no real image needs to be formed on the
detector plane. The nearly isotropic emission of fluorescence excited at
the focal spot allows for new detection schemes that efficiently funnel
all attainable photons to detector(s). We previously showed [Combs,
C.A., et al. (2007) Optimization of *multiphoton* excitation microscopy
by total emission detection using a parabolic light reflector. J.
Microsc. 228, 330-337] that parabolic mirrors and condensers could be
combined to collect the totality of solid angle around the excitation
spot for tissue blocks, leading to ?8-fold signal gain. Using a similar
approach, we have developed an in vivo total emission detection (epiTED)
instrument modified to make noncontact images from outside of living
tissue. Simulations suggest that a ?4-fold enhancement may be possible
(much larger with lower NA objectives than the 0.95 NA used here) with
this approach, depending on objective characteristics, imaging depth and
the characteristics of the sample being imaged. In our initial
prototype, 2-fold improvements were demonstrated in the mouse brain and
skeletal muscle as well as the rat kidney, using a variety of
fluorophores and no compromise of spatial resolution. These results show
this epiTED prototype effectively doubles emission signal in vivo; thus,
it will maintain the image signal-to-noise ratio at two times the scan
rate or enable full scan rate at approximately 30% reduced laser power
(to minimize photo-damage).

Published 2010. This article is a US Government work and is in the
public domain in the USA.

Enhancing collection efficiency in large field of view *multiphoton*
microscopy. <http://www.ncbi.nlm.nih.gov/pubmed/21118215>

McMullen JD, Kwan AC, Williams RM, *Zipfel* WR.

J Microsc. 2011 Feb;241(2):119-24. doi: 10.1111/j.1365-2818.2010.03419.x.

PMID:
    21118215


Many *multiphoton* imaging applications would benefit from a larger
field of view; however, large field of views (>mm) require low
magnification objectives which have low light collection efficiencies.
We demonstrate a light collection system mounted on a low magnification
objective that increases fluorescence collection by as much as 20-fold
in scattering tissues. This peripheral detector results in an effective
numerical aperture of collection >0.8 with a 3-4 mm field of view.

PMID:
    21118215


On 10/24/2014 4:41 PM, Quoc Thang Nguyen wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear List members,
>
> We would like to choose a dry objective lens (x20 to x40) for multiphoton microscopy with a working distance of at least 0.6 mm, 1-2 mm being ideal. Good NA and decent transmittance in NIR (we’ll excite mostly between 800 nm and 940 nm) are required.
>
> Do you have an opinion on the following lenses (in the order of WD)?
>
> ZEISS Plan-Achromat 20X
> NA 0.75
> WD 0.6 mm (a tad short)
> NIKON CFI Super (5) Fluor 20
> NA 0.75
> WD: 1.00 mm
> NIKON N20X-PF - 20X Plan Fluorite Imaging Objective
> NA: 0.50
> WD: 2.1 mm
> NIKON CFI Plan Fluor 20X
> NA 0.50
> WD: 3.00 mm
> OLYMPUS LUCPLFNL 40x
> NA: 0.6
> WD 2.7-4 mm
>
> Thanks for the information.
>
> Best regards to all,
>
> Quoc
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Peter Rupprecht]

Another way to optimize the
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This might be even further off-topic ....

Another way to optimize the fluorescence detection in two photon microscopy is using a ring of large light fibers (Engelbrecht, Goebel, Helmchen):


Optics InfoBase: Optics Express - Enhanced fluorescence signal in nonlinear microscopy through supplementary fiber-optic light collection


And I've seen a paper describing the method of simply putting a dichroic mirror under the sample, such that it reflects the fluorescence photons (if the sample is not too thick.

Have you tested or are you using any of these methods, or seen other ideas how to improve fluorescence detection in 2p microscopy? I've allways been bothered by the amount of useful photons that is being generated and lost in 2P microscopy, but I'm not sure how good the epiTED and the fiber optics methods work, especially for higher NAs, and whether they are worth the effort.

Peter