Hello again,

thanks for the really helpful replies on my 
query. Concerning the test slide, I'll try to get 
some fish scales from the colleagues at Zoology or from some shop...

Guy, I don't mind if dipoles are invoked, as long 
as it is explained why they are there in the 
first place. The Cox and Kable review is a fine 
example how to do it right, thanks for giving the 
reference. There were more enlightening moments when reading through.
A nice surprise was that Springer is giving away 
the pdf of this very chapter of the book as free 
sample. (At this site, if anybody cares: 
http://www.springer.com/humana+press/molecular%2C+cell+and+stem+cell+biology/book/978-1-58829-157-8)

Concerning the moss, I might have to wait for spring though :-)

Thanks again

Steffen


At 07:48 31.01.2009, you wrote:
>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
>______________________________________________
>Phone +61 2 9351 3176     Fax +61 2 9351 7682
>Mobile 0413 281 861
>______________________________________________
>      http://www.guycox.net
>-----Original Message-----
>From: Confocal Microscopy List 
>[mailto:[log in to unmask]] On Behalf Of Steffen Dietzel
>Sent: Saturday, 31 January 2009 5:19 AM
>To: [log in to unmask]
>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
>
>--
>---------------------------------------------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle 
>Medizin (WBex) Head of light microscopy
>
>Mail room (for letters etc.):
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