Sweet! Nice find, reading now!!

C


Caroline Miller
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On Feb 6, 2009, at 11:06 AM, Steffen Dietzel wrote:

> 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
>>
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