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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 [log in to unmask]:
> *****
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> 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