Dear James,
A week ago I wrote what I thought was a perhaps excessively long and
overly dense message in reply to Theresa's initial query, then I thought I
should sleep on it before sending it, and got distracted by other things.
I guess you may well have used that whole week composing yours ;-) and
reading it just now makes the temptation of sending mine irresistible. I am
largely in agreement with you about the need to change mental habits in this
field, and hope that the emphasis on various matters in my message below is
sufficiently different from yours to make a distinct contribution to this
very important discussion. Your analysis of pile-up effects goes well beyond
anything I have ever looked at. However, in line with Theresa's initial
question, I would say that, while I agree with you that the best strategy
for collecting "native data" is no strategy at all, this isn't the case when
collecting data for phasing. In that case one needs to go back and consider
how to measure accurate differences of intensities, not just accurate
intensities on their own. That is another subject, on which I was going to
follow up so as to fully answer Theresa's message - but perhaps that should
come in another installment!
With best wishes,
Gerard.
--
On Tue, May 07, 2013 at 12:04:33AM +0100, Theresa Hsu wrote:
Dear crystallographers
Is there a good source/review/software to obtain tips for good data
collection strategy using PILATUS detectors at synchrotron? Do we need to
collect sweeps of high and low resolution data separately? For anomalous
phasing (MAD), does the order of wavelengths used affect structure solution
or limit radiation damage?
Thank you.
Theresa
--
Dear Theresa,
You have had several excellent replies to your question. Perhaps I
could venture to add a few more comments, remarks and suggestions, which can
be summarised as follows: with a Pilatus, (1) use fine slicing, (2) use
strategies combining low exposure with high multiplicity, and (3) use XDS!
As the use of Pilatus detectors has spread widely, it has been rather
puzzling to come across so many instances when these detectors are misused,
sometimes on the basis of explicit expert advice that is simply misguided. A
typical example will be to see images collected on a Pilatus 6M with an
image width of 1 degree and an exposure time of 1 second. When you see this,
you know that there is some erroneous thinking (or habit) behind it.
When talking to various users who have ended up with such datasets, and
with people who advocate this kind of strategy, it seems clear that a number
of irrational concerns about fine-slicing and low-exposure+high-multiplicity
strategies have tended to override published rational arguments in favour of
those strategies: there is a fear that if the images being collected do not
show spots discernible by the naked eye to the resolution limit that is
being aimed for, the integration software will then somehow not be able to
find those spots in order to integrate them, and the final data resolution
will be lower than expected. Perhaps this may be of concern in relation with
the use of some integration programs, but if you use XDS, which implements a
full 3D approach to image integration, this is simply not the case: XDS will
collect all the counts belonging to a given reflection, whether those counts
are all from a spot on a single 1-degree image exposed for 1 second, or from
10 consecutive images of 0.1 degree width exposed for 0.1 second each, or
from 100 images obtained by grouping together the same 10 images as
previously collected in 10 successive passes with a 10-fold attenuated beam.
The hallmark of the Pilatus detector is to lead to equivalent signal/noise
ratios for the last two ways of measuring that reflection, because it is a
photon counter and has zero readout noise: therefore the combination
Pilatus+XDS is a powerful one.
What is different between these three strategies, however, is the
quality of the overall dataset they will produce. There is nothing new in
what I am describing below: it is all in the references that Bob Sweet gave
you in his reply, or is an obvious consequence of what is found in these
references.
In case 1 (1-degree, 1 second - "coarse slicing") you would presumably
also be (mis-)advised to use a strategy aiming at collecting a complete
dataset in the minimum number of images. These strategies used to make sense
in the days of films, of image plates, and even of CCDs because of the image
readout noise, but they have no place any longer in the context of Pilatus
detectors. First of all, using 1-degree image widths can only degrade the
precision with which 2D spots on images are lifted to 3D reciprocal space
for indexing, and hence worsen the quality of that indexing and therefore
the accuracy with which the spot locations will be predicted (unless you
carefully "post-refine") - then the integration step perhaps does need to
"hunt" for those spots locally, and needs them to be somewhat visible.
Secondly, 1 degree is usually greater than the angular width of a typical
reflection: the integration process will therefore pick up more background
noise (variance) than it would have done with a smaller image width.
Thirdly, by collecting only enough images to reach completeness you will
have substantial radiation damage in your late images compared to the early
ones (if you don't, it means you have under-exposed your crystal) and will
therefore end up with internal inconsistencies in your dataset, as well as
perhaps some extra, spurious anisotropy of diffraction limits as a result of
having to impose increasingly stringent resolution cut-offs in the later
images. This will affect the internal scaling of that dataset and the final
quality of the merged data.
In case 2 (0.1 degree, 0.1 second - "fine slicing") you will have a
more precise sampling of the 3D shape of each spot, hence more accurate
indexing and prediction of spot positions if you use a genuinely 3D
integration program like XDS. Thanks to that increased precision, spots can
be integrated "blind", even if they are not terribly visible in the images,
and the same number of photons will be collected with no penalty in terms of
noise level, thanks to the photon-counting noiseless-readout nature of the
Pilatus detector. An improvement will be that the finely sampled 3D shape of
the spots will be used by XDS to minimise the impact of background variance
on the integrated intensities. On the other hand, the differential radiation
damage between early and late images will still be the same as in case 1 if
you have chosen one of those old-style strategies (and associated beam
intensity setting) that aim at just about exhausting the useful lifetime of
the crystal by the time you reach completeness.
In case 3 (like case 2, but collecting n times more images with an
n-fold attenuated beam once you have collected a few "characterisation
images" without that attenuation to carry out the initial indexing) you
still have the two advantages of case 2 (the same total number of photons
will be picked up by XDS, even if the individual images are now so weak that
you can't see anything) but you are spreading the radiation damage so thinly
over multiple successive complete datasets that you can choose to later
apply a cut-off on image number at the processing stage, when the statistics
tell you that diffraction quality has become degraded beyond some critical
level. This is much preferable to having to apply different resolution
cut-offs to different images towards the end of a barely complete dataset,
as in cases 1 and 2. The impact of radiation damage will be quite smoothly
and uniformly distributed across the final unique reflections, and your
scaling problems (as well as any spurious anisotropy in your diffraction
limits) will be minimised.
This is becoming quite a long message: you can see why I included a
summary of it at the beginning! Returning to it for a conclusion: Pilatus
detectors, fine-slicing with low-exposure and high-multiplicity strategies,
and XDS are a unique winning combination. If fears that another integration
program may not perform as well as XDS on fine-sliced data make you feel
tempted to revert to old-fashioned strategies (case 1) because it supposedly
makes no difference: resist the temptation! Switch to those Pilatus-adapted
strategies and to XDS, and enjoy the very real difference in the results!
With best wishes,
Gerard
and colleagues at Global Phasing.
--
On Tue, May 07, 2013 at 12:04:33AM +0100, Theresa Hsu wrote:
Dear crystallographers
Is there a good source/review/software to obtain tips for good data
collection strategy using PILATUS detectors at synchrotron? Do we need to
collect sweeps of high and low resolution data separately? For anomalous
phasing (MAD), does the order of wavelengths used affect structure solution
or limit radiation damage?
Thank you.
Theresa
