http://confocal-microscopy-list.275.s1.nabble.com/BioRad-MRC-600-scan-generator-card-tp2232860p2285935.html
query. Concerning the test slide, I'll try to get
first place. The Cox and Kable review is a fine
reference. There were more enlightening moments when reading through.
sample. (At this site, if anybody cares:
>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:
[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
>
>--
>---------------------------------------------------------------------------------------------------
>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.):
>Marchioninistr. 15, D-81377 München
>
>Building location and address for courier, parcel services etc:
>Marchioninistr. 27, D-81377 München (Großhadern)