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
