SHG with LSM 510 NLO system

[Connla Edwards]

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Hi list,

We are looking into enabling Second-harmonic generation (SHG) imaging on our LSM 510 NLO system.  Zeiss tell us that it is not possible to fit the required hardware for forward SHG detection with the current stand: Axioskop FS.

Is it possible to improvise with our current stand, and is it worth trying?  What other cost effective solutions would you guys suggest?  Any input or advice is very much appreciated.

Kind regards,

Connla Edwards
Imaging Specialist / Manager
CLICK – Center for Live Imaging of Cells at Karolinska Institutet
Medical Biochemistry & Biophysics (MBB)
[hidden email], Cell: [phone], URL: www.molneuro.mbb.ki.se/click/

[manoj mathew]

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Dear Connla,

SHG due to phase matching conditions travels predominantly in the forward
direction.

Does your confocal system come with a Transmitted Light Detector (TPMT) for
bright field/DIC imaging?

To start with you could simply try putting a notch filter at the SHG
wavelength (if excitation is at 800nm you could use a 400/10 notch filter)
in the transmitted light path.  Then get the condenser aligned for Kohler
illumination and open the field and condenser diaphragms completely. Remove
any ND, GIF or other filters in the transmitted light path.

With multiphoton excitation ON try imaging with the TPMT. If you have a
strong SHG emitting sample like starch or collagen, and your transmitted
light path is well aligned you should see some SHG signal on the TPMT
images.

If this works, you could consider the following upgrades:

1. A high NA condenser (ideally the collection lens should have an NA equal
to better than the excitation objective NA for optimal SHG imaging. The
regular condensers have an NA of about 0.5 which might be low for SHG
imaging).

2. Replace the TPMT with a more sensitive one (if possible)

During my Ph.D. at ICFO, Barcelona we upgraded a Nikon C1 Si confocal to
include SHG imaging by making use of the transmitted light path and a 1.4NA
oil immersion condenser on the Nikon Tie. We got pretty good SHG images.

Do feel free to contact me separately by email or phone if you might need
more info.

Best Regards,
Manoj

--
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Facility Incharge
Central Imaging and Flow Cytometry Facility (CIFF)
Centre for Cellular and Molecular Platforms (CCAMP)
National Centre for Biological Sciences (NCBS)
GKVK Post, Bellary Road
Bangalore-560065, India
Ph. [phone] <080671%2076277>
Mob. [phone] <098867%2089049>
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On Wed, Sep 6, 2017 at 7:05 PM, Connla Edwards <[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.
> *****
>
> Hi list,
>
> We are looking into enabling Second-harmonic generation (SHG) imaging on
> our LSM 510 NLO system.  Zeiss tell us that it is not possible to fit the
> required hardware for forward SHG detection with the current stand:
> Axioskop FS.
>
> Is it possible to improvise with our current stand, and is it worth
> trying?  What other cost effective solutions would you guys suggest?  Any
> input or advice is very much appreciated.
>
> Kind regards,
>
> Connla Edwards
> Imaging Specialist / Manager
> CLICK – Center for Live Imaging of Cells at Karolinska Institutet
> Medical Biochemistry & Biophysics (MBB)
> [hidden email], Cell: [phone], URL:
> www.molneuro.mbb.ki.se/click/
>

[Zdenek Svindrych-2]

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Hi Connla,
have you tried backward SHG? It's cost effective (you don't need any extra
hardware, maybe just a suitable bandpass filter if you're using external non
-descanned detectors). And it usually works fine.

Of course, SHG is forward-directed, but most (biological) samples are not
absolutely clear and transparent, and some of the SHG gets scattered back to
the objective lens. Given the higher sensitivity of the 'backward' detectors
(compared to the transmitted light PMT) you can get very good results.

If you still want to detect the forward SHG, a not-so-crazy idea might be to
place a mirror (better a cold mirror or a long-pass interference filter)
somewhere within (or below, in your case of upright microscope) the
condenser to reflect the light back into the objective and to the internal
or non-descanned detectors. If you want to be even more close to the 'ideal'
forward SHG detection scheme, you may use a quite-high-NA condenser and
underfill the objective on excitation (this way you achieve the condition of
higher detection NA than excitation NA, the Holy Grail of SHG detection).
But beware: I have not tried this myself, nor have I seen this approach
working anywhere! Good luck!

Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
Department of Biology,University of Virginia
409 McCormick Rd, Charlottesville, VA-22904
http://www.kcci.virginia.edu/
tel: [phone]

---------- Původní e-mail ----------
Od: Connla Edwards <[hidden email]>
Komu: [hidden email]
Datum: 7. 9. 2017 5:23:05
Předmět: SHG with LSM 510 NLO system
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Hi list,

We are looking into enabling Second-harmonic generation (SHG) imaging on our
LSM 510 NLO system. Zeiss tell us that it is not possible to fit the
required hardware for forward SHG detection with the current stand: Axioskop
FS.

Is it possible to improvise with our current stand, and is it worth trying?
What other cost effective solutions would you guys suggest? Any input or
advice is very much appreciated.

Kind regards,

Connla Edwards
Imaging Specialist / Manager
CLICK – Center for Live Imaging of Cells at Karolinska Institutet
Medical Biochemistry & Biophysics (MBB)
[hidden email], Cell: [phone], URL: www.molneuro.mbb.ki.se/
click/
"

[Chris O'Connell]

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We've done backward SHG with our 510 for quite a while, exciting with 850 nm and using an emission bandpass filter at 425 in front of the NDDs.  Works OK.

Chris

[Kate Luby-Phelps]

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We used to do forward SHG routinely on our old LSM510 NLO set up exactly the way Manoj described. We got a special 25mm dia. filter  from Chroma to block the excitation light and pass 400 to 450 nm light, which we placed in the transmitted lightpath where the filter holder is near the polarizer. For many samples, especially collagen in tissue sections, the % SHG that goes forward is so high that we got away with just using the garden variety trans detector and 0.55 NA condenser. It didn't work so well with collagen gels. And keeping stray light to an absolute minimum was necessary - had to cover the scope and even the monitors had to be turned off. However, that system was on an inverted stand. I believe yours is upright? So there may be no convenient holder to put the Chroma filter in the transmitted lightpath. Even so, we have made it work on an AxioExaminer just by placing the filter below the stage on top of the field diaphragm.

Kate

[Steffen Dietzel]

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Dear Connla, dear Zdenk,

in my experience with mammalian soft tissues, backward scattered SHG
works ok for collagen, but for myosin only a small part is scattered
back to the Epi-Detectors. See here for a relative comparison of forward
and backward:

https://commons.wikimedia.org/wiki/File:Forward-SHGred-backwardSHGgreen-Collagen-Myosin-860nm-150um.jpg

In absolute terms the forward signal is much stronger for both. So this
may depend very much on the structure you want to look at (You didn't say).


The idea with the mirror actually works quite well, for SHG and THG. We
have done this a few years ago and also published it. By putting the
sample directly on top of the mirror, we roughly got a 10x signal
improvement compared to normal back scattered SHG:

J Biomed Opt. 2010 Mar-Apr;15(2):026017. doi: 10.1117/1.3374337.
Signal improvement in multiphoton microscopy by reflection with simple
mirrors near the sample.
Rehberg M, Krombach F, Pohl U, Dietzel S.

This is the link to the journal's web page:

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-15/issue-02/026017/Signal-improvement-in-multiphoton-microscopy-by-reflection-with-simple-mirrors/10.1117/1.3374337.full?SSO=1

Let me know if you should have problems getting access to it.

Steffen


Am 07.09.2017 um 15:32 schrieb [hidden email]:

> *****
> 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.
> *****
>
> Hi Connla,
> have you tried backward SHG? It's cost effective (you don't need any extra
> hardware, maybe just a suitable bandpass filter if you're using external non
> -descanned detectors). And it usually works fine.
>
> Of course, SHG is forward-directed, but most (biological) samples are not
> absolutely clear and transparent, and some of the SHG gets scattered back to
> the objective lens. Given the higher sensitivity of the 'backward' detectors
> (compared to the transmitted light PMT) you can get very good results.
>
> If you still want to detect the forward SHG, a not-so-crazy idea might be to
> place a mirror (better a cold mirror or a long-pass interference filter)
> somewhere within (or below, in your case of upright microscope) the
> condenser to reflect the light back into the objective and to the internal
> or non-descanned detectors. If you want to be even more close to the 'ideal'
> forward SHG detection scheme, you may use a quite-high-NA condenser and
> underfill the objective on excitation (this way you achieve the condition of
> higher detection NA than excitation NA, the Holy Grail of SHG detection).
> But beware: I have not tried this myself, nor have I seen this approach
> working anywhere! Good luck!
>
> Best, zdenek

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Connla Edwards]

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Hello List,



Thank you all who responded with such valuable information.  We will
investigate the options you have put forward.


Cheers,

Connla


On Fri, Sep 8, 2017 at 3:05 PM, Kate Luby-Phelps <
[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.
> *****
>
> We used to do forward SHG routinely on our old LSM510 NLO set up exactly
> the way Manoj described. We got a special 25mm dia. filter  from Chroma to
> block the excitation light and pass 400 to 450 nm light, which we placed in
> the transmitted lightpath where the filter holder is near the polarizer.
> For many samples, especially collagen in tissue sections, the % SHG that
> goes forward is so high that we got away with just using the garden variety
> trans detector and 0.55 NA condenser. It didn't work so well with collagen
> gels. And keeping stray light to an absolute minimum was necessary - had to
> cover the scope and even the monitors had to be turned off. However, that
> system was on an inverted stand. I believe yours is upright? So there may
> be no convenient holder to put the Chroma filter in the transmitted
> lightpath. Even so, we have made it work on an AxioExaminer just by placing
> the filter below the stage on top of the field diaphragm.
>
> Kate
>

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