Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/BioRad-MRC-600-scan-generator-card-tp2232860p2249034.html
OK, it's kind of hard not to invoke dipoles here. I'm a biologist not a physicist but in the end we're talking about a physical process.
If a sample contains a few small objects the direction of the SHG signal will be very dependent on the orientation of the objects. In Guy Cox & Eleanor Kable, 2006. Second Harmonic Imaging of Collagen. In: D.J. Taatjes and B.T. Mossman (Eds.), Cell Imaging Techniques. (Methods in Molecular Biology, Volume 319) Humana Press, Totowa, NJ, pages 15-35, Figure 3 A-D there is is a diagram of this (borrowed by permission from Sunney Xie) which illustrates the way the signal will go. (It was originally drawn for CARS microscopy but the physical considerations are the same - the source is Ji-Xin Cheng, Y. Kevin Jia, Gengfeng Zheng & X. Sunney Xie, 2002. Laser-Scanning Coherent Anti-Stokes Raman Scattering Microscopy and Applications to Cell Biology. Biophysical Journal 83, 502-509).
Once you get a lot of dipoles (molecules) together the propagation tends to be forward, as shown in the last part (E, F) of the above figure (mine now not Sunney's!), and also in Chapter 8 of my book (below). This is, as you say, because back-propagation will not be in phase but forward propagation will. This does NOT mean that energy is lost in the sample, just that it goes forwards. When you get destructive and constructive interference (as in any diffracting specimen) the energy 'lost' in destructive interference equals that 'gained' in the constructive interference - it is just a redirection.
These diagrams are all based on fairly low NA excitation - if I understand it right (and remember I'm just a biologist) the 'hollow-cone' bit comes about because in a very high NA system the phases of the incoming beam get a bit scrambled in the focussed spot. The only practical consequence of this is to make sure that you are collecting the transmitted signal with at least as high an NA as the objective - ie use an oil-immersion condenser.
So there are two ways you can get back-propagation of the SHG signal. If you have many dipoles in line side to side (but not in front or behind) the signal will go equally forwards and backwards. Or you can have a 'bulk' specimen, giving a very strong signal which would normally propagate forwards, but which scatters light so much that the signal will get diverted in all directions.
I hope this helps. I'm sure any unidentified moss from a Munich wall will work as well as a Sydney one. The interesting bit with that was to try to get 3D images of TPF of chloroplasts as well as SHG of starch, since PC Cheng showed that chloroplasts are very easily damaged by two-photon excitation. The point of the exercise was to show we could get a full 3D dataset in TPF & SHG without damaging the chloroplasts.
As to a standard sample, I've mentioned before that Bio-Rad used to supply a starch-grain sample with their MRC 500 & 600 confocals and since these were very common microscopes I'm sure there must be lots of those slides about. Otherwise, contact your local histology depafrtment and get a slide of skin, or tendon, which will have a lot of collagen in it.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
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http://www.guycox.net-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Steffen Dietzel
Sent: Saturday, 31 January 2009 5:19 AM
To:
[hidden email]
Subject: Second and Third Harmonic Generation - 3D distribution and test slides
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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