Re: Second and Third Harmonic Generation - 3D distribution and test slides

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/BioRad-MRC-600-scan-generator-card-tp2232860p2246997.html

SHG is a coherent process; this means that you are almost making the sample lase sort-of.  As a result, the output tends to go in the direction of the input.  This means that the strongest SHG signal is in the forward direction, i.e. traveling with the source beam.  There is a very weak naturally produced backwards-travelling signal, but this depends on some weird conditions and requires sensitive detection and/or a relatively noiseless scenario to detect.  In thick samples, the forward propagating signal can actually bounce off the lower layers of the sample and come back at you.  This is backscattered SHG and can be picked up in the epi direction.  The strength of it depends on the thickness and scattering characteristics of your sample.  Usually though, since SHG is higher-frequency it tends to scatter fairly well.
Our lab has tried a few tricks for SHG;
-Rotate your laser polarization; because it is a coherent process it is very polarization sensitive and you will find that your image brightness (or portions of your image) will increase or decrease as you change the polarization angle.
-A thick sample will scatter enough forward SHG in the backward direction (backscatter) that most epi microscopes can pick it up
-For thin samples you may want to try putting a reflective dichroic filter underneath your sample (pass the main beam and reflect SHG) to get more signal
The classic sample that works well is a rat tail slice.

Hope this helps!

Craig

On Fri, Jan 30, 2009 at 11:18 AM, Steffen Dietzel <[hidden email]> wrote:

Dear all,

I am trying to get a better understanding of Higher Harmonic Generation.

From what I have read and experienced so far, the forward second Harmonic Generation (SHG) signal is in most cases stronger than the backward signal.

Is there a theory or investigaton about the 3D-distribution, i.e. what "forward" and particularly "backward" acutally mean? I found one paper for forward SHG (and THG) that explains that "forward" is acutally not exactly forward but the SHG signal is distributed as a hollow cone, with nothing at the center (Moreaux et al., 2001, http://www.ncbi.nlm.nih.gov/pubmed/11222317). But I didn't find anything for the backward signal.

I heard and read several opinions, some of which are mutually exclusive.
- backward SHG is just forward SHG signal which is scattered back.
- Some objects produce more backward SHG signal than others (relative to the forward signal)
- "backward" is not exactly backward but goes away to the side, at some angle to the optical axis (hollow cone, as for forward)
- Forward Third Harmonic Generation (THG) signal is distributed also as a hollow cone, but tighter (in the Moreaux-Paper)
- THG is not oriented, goes in all directions equally.

I'd be glad if people could comment on these points.
I guess good reviews on these subjects would also help. The problem is that many of such articles use tech speak which might be ok for physicists but partly incomprehensible for others if they use stuff like "cross-section", "dipoles" or "vector electric field" without explaining them.

More of academic interest: I found a statement that, at first, SHG is produced equally towards all sides (or at least more directions) but then, in a second step, wave interference nihilates it except for the forward direction. However, if there is destructive interference of light, the energy must stay somewhere. Is the statement that no energy deposition occurs in the sample thus really true? (Assuming that there is no regular absorbtion and autofluorescence).

Another one out of academic interest: Articles often write something like "Higher harmonic generation, including SHG and THG" - Is there anything but these two? If we could get a >1600 nm laser, would we start to see Fourth Harmonic Generation?

Also, has anybody an idea for good SHG/THG test slides with reproducible signals? Inspired by papers of  Guy Cox, I have tried microtome sections of fresh potatoes which contain a lot of SHG signal-generating starch granules, but the granules vary a lot in size and signal. (I still have to see whether I can find the equivalent of an 'unidentified moss species from a Sydney wall' in a Munich winter :-)  ). Urea crystals do not work well with water dipping objectives. Collagen matrix sort of worked if we stayed above the minimum laser power to generate a signal and below the point where we fry the matrix, the corridor is not too wide. No ideas for THG tests so far.

Thanks for any help

Steffen

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Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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