BioRad MRC 600 scan generator card

[Darren Korber]

Hi We have an old BioRad MRC 600 that is still (almost, anyway) in good working order. Unfortunately, it seems that the scan generator card has developed a problem and so we need to replace it and see if this fixes things. Anyone still have one of these old units or parts, and would they be willing to provide this card? Thanks Darren Korber

[Steffen Dietzel]

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)

Phone: [phone]
Fax-to-email:   +49/89/2180-9976509
skype: steffendietzel
e-mail: dietzel at lmu dot de

[Jerry Sedgewick-2]

Hello Steffen,

I can't comment intelligently on the workings of SHG and THG, but I can
tell you what we use as a consistent sample for forward scatter SHG.  We
have mounted zebrafish scales in a hardening mounting media on a
microscope slide and we start every session by imaging from this.

Jerry


Steffen Dietzel 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
>

--
Jerry (Gerald) Sedgewick
Program Director, Biomedical Image Processing Lab (BIPL)
Department of Neuroscience, University of Minnesota
312 Church St. SE, 1-205 Hasselmo Hall
Minneapolis, MN  55455
[phone]
[hidden email]
http://www.bipl.umn.edu
Author: "Scientific Imaging with Photoshop: Methods, Measurement and Output."

Rawlight.com (dba Sedgewick Initiatives)
965 Cromwell Avenue
Saint Paul, MN  55114
[hidden email]
[phone]
http://www.quickphotoshop.com
http://www.heartFROMstone.com
http://www.rawlight.com




--- Get FREE High Speed Internet from USFamily.Net! -- http://www.usfamily.net/mkt-freepromo.html ---

[Craig Brideau]

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

--
---------------------------------------------------------------------------------------------------
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)

Phone: [phone]
Fax-to-email:   +49/89/2180-9976509
skype: steffendietzel
e-mail: dietzel at lmu dot de

[Guy Cox]

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 [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     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)

Phone: [phone]
Fax-to-email:   +49/89/2180-9976509
skype: steffendietzel
e-mail: dietzel at lmu dot de

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Checked by AVG.
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[Steffen Dietzel]

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 [phone]     Fax [phone]
>Mobile [phone]
>______________________________________________
>      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)

[Caroline Miller]

Sweet! Nice find, reading now!!

C


Caroline Miller
Co-Manager
J David Gladstone Institutes Histology and Microscopy Core
1650 Owens St
San Francisco
CA 94158

Tel: [phone]
Fax: [phone]

http://www.gladstone.ucsf.edu/gladstone/site/histology/
[hidden email]





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 [phone]     Fax [phone]
>> Mobile [phone]
>> ______________________________________________
>>     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:
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