Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Quoting Jacob Keller j-kell...@md.northwestern.edu: Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? It is certainly not correct to state that there is no anomalous scattering in elemental Se. There is anomalous scattering: the atomic form factors f' and f have the specific wavelength-dependence, which can be measured from the diffraction data (by collecting data at different wavelengths); you can collect a fluorescence scan over the absorption edge etc. However, because there is only one type of scatterer (the f' + if for all atoms are the same), Friedel's law remains valid, i.e. I(+h) and I(-h) remain the same. And even this is only true as long as we consider that the atoms are spherical and neglect anisotropy of anomalous scattering etc. Marc I beg to differ again with regard to our selenium crystal: there is a normal diffraction pattern arising from the unbound [majority of] electrons (imagine the crystal below the K-edge, for example--no resonant scattering, right?), but then there is also another signal arising from the resonant scattering, which has a definite phase lag with respect to the elastically-scattered wave. Is there something I am missing?
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Quoting Jacob Keller j-kell...@md.northwestern.edu: Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption--fluorescence? I doubt that this is a correct interpretation. It is f which is related to absorption (and therefore to fluorescence) not f' ! In fact f' can be positive, even if there is absorption (and fluorescence). Examples: the f' factors of C, O, S, Cl and most other lighter elements are positive at the Cu K-alpha wavelength, but they are still absorbing. The optical theorem relates \mu, the macroscopic absorption coefficient, to f, NOT to f' ! The amount that any material absorbs is in no way related to the f' factors of the atoms of which it is build up. But it is directly related to their f factors. When you collect a fluorescence scan, you get a quantity which is directly related to f and NOT to f' (the raw scan resembles already very much the spectral curve of f). To get f', you have to perform a Kramers-Kronig transform. The macroscopic counterpart of f' is dispersion, i.e. a change of phase velocity. Marc
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Quoting Jacob Keller j-kell...@md.northwestern.edu: Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? It is certainly not correct to state that there is no anomalous scattering in elemental Se. There is anomalous scattering: the atomic form factors f' and f have the specific wavelength-dependence, which can be measured from the diffraction data (by collecting data at different wavelengths); you can collect a fluorescence scan over the absorption edge etc. However, because there is only one type of scatterer (the f' + if for all atoms are the same), Friedel's law remains valid, i.e. I(+h) and I(-h) remain the same. And even this is only true as long as we consider that the atoms are spherical and neglect anisotropy of anomalous scattering etc. Marc
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? By the way, while we're on the topic of comparing uv-vis fluorescence to x-ray fluorescence, does anybody know of an example of the use of FRET in x-ray fluorescence? I cannot think, off hand, of an application for such, but theoretically it could be done easily with two types of heavy atoms, such as a Se-met and some appropriate acceptor. Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 23, 2009 8:59 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence Dirk Kostrewa wrote: yes, this is certainly true for real fluorescence effects. But the anomalous scattering can be best thought of as a resonance phenomenon without any frequency change, and as such, it has a distinct phase relationship to the elastically scattered photon and does have an effect on the intensities (which, I think, was the background of the original question?). But for the lighter atoms in biological macromolecules, where in a typical experiment the measurement frequency is far away from any resonance frequency, this effect can be neglected. This leads me to my follow-up question to the experts: why is the resonance effect anomalous scattering measured by a fluorescence scan that should have all the effects mentioned by James? Don't we get as a result a mixture of signals from resonance (i.e. anomalous) and from absorption-emission (i.e. fluorescence) effects? Fluorescent photon emission happens well after the incident photon has passed, so anomalous scattering is only indirectly related to fluorescence. The relationship is that absorption induces a phase shift in scattering (this is the anomalous scattering effect), but it also induces an electronic transition in the atom, leaving a core hole or vacant orbital near the nucleus. The filling of this core hole will generate a fluorescent photon (some fixed fraction of the time), and this allows us to equate the intensity of observed fluorescence to the number of core holes produced and therefore to the absorption cross section of the atom. In actual fact, the MAD scan we do before a MAD/SAD experiment is not a fluorescence spectrum, but rather an absorption spectrum using fluorescence as a tally. A fluorescence spectrum would have the energy of the fluorescent photon on the x-axis. (Bob Sweet has corrected me several times for getting that wrong). As for the connection between absorption and anomalous scattering, I tend to think of this in the classical picture. Scattering lags the incident beam by 90 degrees because a simple harmonic oscillator driven at frequencies much higher than resonance lags behind the force upon it. An oscillator driven at resonance will move 180 degrees out-of-phase with the driving force. You can verify this yourself by playing with a weight tied to the end of a rubber band. Another way to think about it is that absorption must create a wave that is 180 degrees out of phase with the incident beam because it reduces the intensity of the incident beam. The details of the physics are much more complicated than this, but this is how I like to remember it. So, as you approach a resonance, some of the electrons in the atom will start absorbing (resonating) and therefore move out-of-phase with the other electrons in the atom (and indeed the other electrons in the crystal). It is this out of sync behavior that reduces the effective occupancy of the atom and also creates an imaginary component to the scattering. This imaginary electron density is hard to accept if you have never taken complex algebra, but the easy way to think about it is to remember than multiplying a complex number by sqrt(-1) changes its phase by 90 degrees. So the imaginary component is really just a mathematical way to represent electrons that are out-of-sync with the majority of electrons in the crystal. Yes, the majority, because a pure selenium crystal has no anomalous scattering (since no atoms lag any other atoms). The imaginary component is what leads to the breakdown of Friedel's law
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption--fluorescence? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:53:27 Jacob Keller wrote: Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption--fluorescence? I am not sure about that because. Let's not confuse correlation with causality. The negative f' is adequately explained by the Kramers-Kronig equation as being a result of the resonance interaction. http://www.rp-photonics.com/kramers_kronig_relations.html The maximum resonance is at the absorption energy, which is also the maximum for the fluorescence. Both effects are because of the match between incident photon energy and the energy required to kick an electron out of its current orbital state. I am uneasy saying that one effect causes the other effect. Ethan Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Friday 24 April 2009 11:53:27 Jacob Keller wrote: Aha, so I have re-invented the wheel! But I never made sense of why f' is negative--this is beautiful! Just to make sure: you are saying that the real part of the anomalous scattering goes negative because those photons are sneaking out of the diffraction pattern through absorption-- fluorescence? I am not sure about that because. Let's not confuse correlation with causality. The negative f' is adequately explained by the Kramers- Kronig equation as being a result of the resonance interaction. http://www.rp-photonics.com/kramers_kronig_relations.html The maximum resonance is at the absorption energy, which is also the maximum for the fluorescence. Both effects are because of the match between incident photon energy and the energy required to kick an electron out of its current orbital state. I am uneasy saying that one effect causes the other effect. Ethan There is a very good technical description in Jens Als-Nielsen's Elements of Modern X-ray Physics in the chapter Resonant Scattering, pg 235 ff. In fact, there is also a good description of the breakdown of Friedel's Law and the MAD experiment in that chapter. I would like to iterate Ethan's comment about resonance. The effects are not anomalous at all, we know very well what is happening: the changes in f', f and mu as a function of energy are all effects of the resonance of the photon energy with transition energy of the electron. So, we really should call it resonance scattering, not anomalous scattering. I have to admit MRD and SRD aren't as euphonic at MAD and SAD and the change will probably never happen. Joe Joseph D. Ferrara, Ph.D. Rigaku Americas Corporation Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: Jacob Keller j-kell...@md.northwestern.edu; CCP4BB@jiscmail.ac.uk Sent: Friday, April 24, 2009 1:40 PM Subject: Re: [ccp4bb] Reason for Neglected X-ray Fluorescence On Friday 24 April 2009 11:28:16 Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Yes. That is why the real component of the scattering factor, f', is negative. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Jacob Keller wrote: Dear Dr. Holton and CCP4BBers, Are you saying that a resonant event is always accompanied by a fluorescence event? no. For example, with selenium only ~59% of the core holes decay by emitting a fluorescent x-ray. The rest by emitting an Auger electron. The latter seldom escape the sample. On the other hand, there are generally a lot more absorbed photons than scattered ones. For Se again at 12680 eV (just above the edge) the ratio is about 120 absorption events for every elastically scattered photon. And since 59% of the absorptions make a fluorescent x-ray, there are about 70 fluorescence events for every resonant event. Anyway, the ratio is definitely not 1:1. If that were true, wouldn't the resonant event end up manifesting as *negative* scattering component from the resonant atom, due to the elimination of an otherwise-scattered photon, this making the resonant atom darker than would be expected? Sort of. I personally like to think of the core electrons as disappearing from the normal scattering as they start to scatter out of phase. However, ALL of the electrons scatter any given photon. Even the anomalously scattering electrons don't really disappear any more than a beat-deaf member of a marching band disappears when they get out of step with the rest of the rank and file, but they do stop contributing to the total effect. Also, in your selenium crystal example, I think there would still be an anomalous signal, because there would always be regular scattering as well as the anomalous effect. Isn't that true? No. There is no anomalous scattering from crystals with only one atom type. That is, Friedel's law holds because the phase lag from every atom is the same. Friedel's law also holds for centrosymmetric crystals, despite any anomalous effects. I suppose you might be able to see the atomic form factor change as the core electrons go out of phase as you approach the absorption edge, and well, okay, technically that is an anomalous scattering effect. But Friedel's law is not broken for elemental crystals nor for centrosymmetric crystals. By the way, while we're on the topic of comparing uv-vis fluorescence to x-ray fluorescence, does anybody know of an example of the use of FRET in x-ray fluorescence? I cannot think, off hand, of an application for such, but theoretically it could be done easily with two types of heavy atoms, such as a Se-met and some appropriate acceptor. Rick Donahue (or health physicist here at ALS) told me a story once where they found a sample of what I think was some metal carbonate that was emitting fluorescent x-rays from the metal, but it was the carbon in the carbonate that was radioactive. One could consider this an example of a transfer of excitation in the x-ray regime, but I'll have to check with Rick to be sure. -James Holton MAD Scientist
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Am 23.04.2009 um 02:31 schrieb James Holton: Fluorescent x-rays have a VERY different wavelength from the incident beam and therefore cannot interact coherently with Bragg- scattered photons, so they contribute to nothing but background. Fluorescence is also a true absorption-reemission process, and must occur from one atom at a time. The core hole lifetime before emission occurs is small, but there is still a random delay before the fluorescent photon is emitted. This means there is essentially no interference between fluorescence events from different atoms. Scattering, on the other hand, occurs from every atom in the crystal simultaneously for each incident photon, and this is why we see interference. yes, this is certainly true for real fluorescence effects. But the anomalous scattering can be best thought of as a resonance phenomenon without any frequency change, and as such, it has a distinct phase relationship to the elastically scattered photon and does have an effect on the intensities (which, I think, was the background of the original question?). But for the lighter atoms in biological macromolecules, where in a typical experiment the measurement frequency is far away from any resonance frequency, this effect can be neglected. This leads me to my follow-up question to the experts: why is the resonance effect anomalous scattering measured by a fluorescence scan that should have all the effects mentioned by James? Don't we get as a result a mixture of signals from resonance (i.e. anomalous) and from absorption-emission (i.e. fluorescence) effects? Best regards, Dirk. *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@lmb.uni-muenchen.de ***
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
James Holton wrote:
marc.schi...@epfl.ch wrote:
The elastically scattered photons (which make up the Bragg peaks) also
do not not retain the momentum of the incident photon.
Although technically true to say that photons traveling in different
directions have different momenta, all elastically scattered photons
have the same wavelength (momentum) as the incident photon. Otherwise,
I would definitely avoid to amalgamate wavelength and momentum, as is
more-or-less suggested in the final part of this statement. Momentum is
a vector quantity, although it is true that the NORM of the momentum
vector of a particle is related to its energy (by the De Broglie
wavelength relation). In X-ray diffraction, the momentum of the
elastically scattered photon does change, while its energy does not. In
X-ray physics, the change in momentum is actually called the momentum
transfer : \vec{Q} = \vec{k'} - \vec{k}. The word says it all.
they would not interfere constructively to form Bragg peaks and they
would be called Compton-scattered photons. The small change in energy
required to preserve wavelength upon a change in direction during
elastic scattering is contributed by the entire crystal as a recoil
phonon. Arthur Compton wrote a paper about this:
http://www.pnas.org/cgi/reprint/9/11/359.pdf
Very interesting paper, but I see no mention of a recoil phonon and I
would be surprised if that is what Compton really meant. No mention
about lattice dynamics (phonons) can be found in this paper. The crystal
is implicitly assumed to be a rigid body. In fact, what the paper nicely
demonstrates is that the conservation of wavelength (i.e. photon energy)
between incident and diffracted rays is achieved in the limiting case
when the total mass of the crystal is very large with respect to the
mass of one photon - a condition which, I presume, is always satisfied
in X-ray crystallography, even when going towards microcrystals.
This is really the same situation as a tennis ball that bounces
(elastically) off the surface of the earth. In principle, we must assume
that some of its energy is transferred to the earth during the
collision. But because the mass of the earth is so vastly superior to
the mass of the tennis ball, the transfer of energy is vanishingly
small. It certainly can not be measured. The change of momentum of the
tennis ball, however, is not negligible and can be measured.
Back to X-ray diffraction, the reciprocal lattice is just a
representation of momentum transfer vectors \vec{Q} = 2\pi \vec{h}. You
may never have thought of it like this, but when we index an X-ray
pattern, we are really measuring the change in momentum of the photons
which were scattered into the various Bragg peaks. But we can not
measure their change in energy, as it is practically zero.
The situation becomes somewhat different if we take into account lattice
dynamics (phonons) as it is now possible to measure the energy transfer
of a scattered X-ray photon upon phonon creation in the crystal. But
these are very difficult measurements (much easier with neutrons) and
are certainly of no relevance for macromolecular X-ray crystallography.
It is anyway called inelastic scattering.
which probably contributed to his Nobel four years later. This is a
classic example of the confusion that can arise from the particle-wave
duality.
It seems to me that the confusion here is between energy and momentum.
--
Marc SCHILTZ http://lcr.epfl.ch
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
For those who are still following this discussion...
Following a comment by James, I clarify my previous statement about
the limiting case when the total mass of the crystal is very large
with respect to the mass of one photon
I meant of course the relativistic mass of one photon [which is given
by h/(\lambda c)]. The rest mass of a photon is of course zero.
A photon of \lambda = 1 Angstroem has a relativistic mass of the order
of 10^{-32} kg. Certainly much smaller than the mass of even a
nano-crystal...
I was really just re-phrasing what Arthur Compton wrote in the quoted
paper [read the sentence just after his equation (9)].
--
Marc SCHILTZ http://lcr.epfl.ch
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Dirk Kostrewa wrote: yes, this is certainly true for real fluorescence effects. But the anomalous scattering can be best thought of as a resonance phenomenon without any frequency change, and as such, it has a distinct phase relationship to the elastically scattered photon and does have an effect on the intensities (which, I think, was the background of the original question?). But for the lighter atoms in biological macromolecules, where in a typical experiment the measurement frequency is far away from any resonance frequency, this effect can be neglected. This leads me to my follow-up question to the experts: why is the resonance effect anomalous scattering measured by a fluorescence scan that should have all the effects mentioned by James? Don't we get as a result a mixture of signals from resonance (i.e. anomalous) and from absorption-emission (i.e. fluorescence) effects? Fluorescent photon emission happens well after the incident photon has passed, so anomalous scattering is only indirectly related to fluorescence. The relationship is that absorption induces a phase shift in scattering (this is the anomalous scattering effect), but it also induces an electronic transition in the atom, leaving a core hole or vacant orbital near the nucleus. The filling of this core hole will generate a fluorescent photon (some fixed fraction of the time), and this allows us to equate the intensity of observed fluorescence to the number of core holes produced and therefore to the absorption cross section of the atom. In actual fact, the MAD scan we do before a MAD/SAD experiment is not a fluorescence spectrum, but rather an absorption spectrum using fluorescence as a tally. A fluorescence spectrum would have the energy of the fluorescent photon on the x-axis. (Bob Sweet has corrected me several times for getting that wrong). As for the connection between absorption and anomalous scattering, I tend to think of this in the classical picture. Scattering lags the incident beam by 90 degrees because a simple harmonic oscillator driven at frequencies much higher than resonance lags behind the force upon it. An oscillator driven at resonance will move 180 degrees out-of-phase with the driving force. You can verify this yourself by playing with a weight tied to the end of a rubber band. Another way to think about it is that absorption must create a wave that is 180 degrees out of phase with the incident beam because it reduces the intensity of the incident beam. The details of the physics are much more complicated than this, but this is how I like to remember it. So, as you approach a resonance, some of the electrons in the atom will start absorbing (resonating) and therefore move out-of-phase with the other electrons in the atom (and indeed the other electrons in the crystal). It is this out of sync behavior that reduces the effective occupancy of the atom and also creates an imaginary component to the scattering. This imaginary electron density is hard to accept if you have never taken complex algebra, but the easy way to think about it is to remember than multiplying a complex number by sqrt(-1) changes its phase by 90 degrees. So the imaginary component is really just a mathematical way to represent electrons that are out-of-sync with the majority of electrons in the crystal. Yes, the majority, because a pure selenium crystal has no anomalous scattering (since no atoms lag any other atoms). The imaginary component is what leads to the breakdown of Friedel's law (which states that the Fourier transform of a real-valued function is centrosymmetric). But all this is really just a fancy way of saying that some of the electrons are out of phase with the rest. Hope this makes sense. -James Holton MAD Scientist Best regards, Dirk. *** Dirk Kostrewa Gene Center, A 5.07 Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail:kostr...@lmb.uni-muenchen.de ***
[ccp4bb] Reason for Neglected X-ray Fluorescence
Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: rui To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, April 22, 2009 11:06 AM Subject: [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Dear Jacob, I think it is because the fluorescence is incoherent, and hence contributes to the background rather than to the diffraction. Congratulations, by the way, for managing to spell fluorescence correctly twice within a short space! Usually it occurs as flourescence, which if pronounced flour essence conjures up the image of a rather unappealing substance. With best wishes, Gerard. -- On Wed, Apr 22, 2009 at 11:23:19AM -0500, Jacob Keller wrote: Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Wednesday 22 April 2009 09:23:19 Jacob Keller wrote: Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Fluorescence is directly proportional to f, so in one sense we do account for it in any calculation that includes the anomalous scattering terms. If you were thinking of direct contribution of the fluorescent X-rays to the measured Bragg peak - that is negligible. Those photons do not retain the momentum vector of the original incident photon, and are emitted in all directions. I.e., they contribute even less to the diffraction image than air-scatter from the direct beam or from the diffracted beam. Ethan Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: rui To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, April 22, 2009 11:06 AM Subject: [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Because the fluorescence comes off in all directions, it makes a significant contribution to the noise as one approaches the resolution limit of the crystals, limiting the maximum resolution attainable. So it would be a good idea to try to reduce it, e.g. by choosing a suitable wavelength. Air scattering increases the noise at lower resolution, which is less serious. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 22 Apr 2009, Gerard Bricogne wrote: Dear Jacob, I think it is because the fluorescence is incoherent, and hence contributes to the background rather than to the diffraction. Congratulations, by the way, for managing to spell fluorescence correctly twice within a short space! Usually it occurs as flourescence, which if pronounced flour essence conjures up the image of a rather unappealing substance. With best wishes, Gerard. -- On Wed, Apr 22, 2009 at 11:23:19AM -0500, Jacob Keller wrote: Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
Quoting Ethan Merritt merr...@u.washington.edu: On Wednesday 22 April 2009 09:23:19 Jacob Keller wrote: Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Fluorescence is directly proportional to f, so in one sense we do account for it in any calculation that includes the anomalous scattering terms. If you were thinking of direct contribution of the fluorescent X-rays to the measured Bragg peak - that is negligible. Those photons do not retain the momentum vector of the original incident photon, and are emitted in all I am not sure whether this is a good explanation. The elastically scattered photons (which make up the Bragg peaks) also do not not retain the momentum of the incident photon. directions. I.e., they contribute even less to the diffraction image than air-scatter from the direct beam or from the diffracted beam. Well, this clearly depends on the sample content and on the X-ray wavelength. There are many examples of data collected at an absorption edge, where fluorescence is the dominating contributor to the background, i.e. it is much larger than air-scatter from the direct beam or from the diffracted beams. For an extreme case, see fig. 4 in Shepard et al.(2000). Acta Cryst. D56, 1288-1303. Marc Ethan Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: rui To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, April 22, 2009 11:06 AM Subject: [ccp4bb] microbatch vs hanging drop Hi, I have a question about the method for crystallization. With traditional hanging drop(24 wells), one slide can also hold for multiple drops but it requires the buffer quite a lot, 600uL? Microbatch can save buffers,only 100uL is required, and also can hold up to three samples in the sitting well. Other than saving the buffer, what's the advantage of microbatch? Which method will be easier to get crystals or no big difference? Thanks for sharing. R -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
On Wednesday 22 April 2009 13:22:41 marc.schi...@epfl.ch wrote: Quoting Ethan Merritt merr...@u.washington.edu: On Wednesday 22 April 2009 09:23:19 Jacob Keller wrote: Hello All, What is the reason that x-ray fluorescence is neglected in our experiments? Obviously it is measureable, as in EXAFS experiments to determine anomalous edges, but should it not play a role in the intensities as well? What am I missing? Fluorescence is directly proportional to f, so in one sense we do account for it in any calculation that includes the anomalous scattering terms. If you were thinking of direct contribution of the fluorescent X-rays to the measured Bragg peak - that is negligible. Those photons do not retain the momentum vector of the original incident photon, and are emitted in all I am not sure whether this is a good explanation. The elastically scattered photons (which make up the Bragg peaks) also do not not retain the momentum of the incident photon. Poorly phrased, I guess. My thought was that elastic scattering from air favors smaller deflection angles, which means more of the photons continue forward to strike the detector than scatter at large angles and miss it. Is this not correct? There is no such bias for the fluorescent photons, so they are as likely to travel back towards the source as towards the detector. Ethan directions. I.e., they contribute even less to the diffraction image than air-scatter from the direct beam or from the diffracted beam. Well, this clearly depends on the sample content and on the X-ray wavelength. There are many examples of data collected at an absorption edge, where fluorescence is the dominating contributor to the background, i.e. it is much larger than air-scatter from the direct beam or from the diffracted beams. For an extreme case, see fig. 4 in Shepard et al.(2000). Acta Cryst. D56, 1288-1303. Marc Ethan Jacob -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] Reason for Neglected X-ray Fluorescence
marc.schi...@epfl.ch wrote: The elastically scattered photons (which make up the Bragg peaks) also do not not retain the momentum of the incident photon. Although technically true to say that photons traveling in different directions have different momenta, all elastically scattered photons have the same wavelength (momentum) as the incident photon. Otherwise, they would not interfere constructively to form Bragg peaks and they would be called Compton-scattered photons. The small change in energy required to preserve wavelength upon a change in direction during elastic scattering is contributed by the entire crystal as a recoil phonon. Arthur Compton wrote a paper about this: http://www.pnas.org/cgi/reprint/9/11/359.pdf which probably contributed to his Nobel four years later. This is a classic example of the confusion that can arise from the particle-wave duality. Fluorescent x-rays have a VERY different wavelength from the incident beam and therefore cannot interact coherently with Bragg-scattered photons, so they contribute to nothing but background. Fluorescence is also a true absorption-reemission process, and must occur from one atom at a time. The core hole lifetime before emission occurs is small, but there is still a random delay before the fluorescent photon is emitted. This means there is essentially no interference between fluorescence events from different atoms. Scattering, on the other hand, occurs from every atom in the crystal simultaneously for each incident photon, and this is why we see interference. -James Holton MAD Scientist
