Dear Frank,
Very good question. You are right: in this statement I used "XDS"
rather metaphorically, in the sense that this grouping together of all the
counts will happen at merging time rather than at integration time. In the
end you will have a counting-statistics contribution to the variance of the
merged measurement that would be the same as if you had collected the total
number of photons in one lot. Of course that variance will incorporate many
other CORRECTions, but essentially you will get a similar final I/sig(I)
value in all these cases. The final measurement produced by multi-pass fine
slicing with low exposure has a good chance of being the most immune to
radiation damage, because you would be able to use zero-dose extrapolation
on the numerous individual spots present in the resulting images, and to
cancel the trend created by radiation damage beyond the simple decrease that
gets modelled by image B-factors. This is a much better idea than to combine
images 360 degrees apart.
I hope this answers the first of your questions. For the second I need
to think a bit more - the drop in raw integrated intensity divided by the
background is an obvious criterion to consider - but you may get suggestions
from many others.
With best wishes,
Gerard.
--
On Thu, May 16, 2013 at 06:25:35PM +0100, Frank von Delft wrote:
> Dear Gerard - thanks, very informative! Two questions:
>
> 1.
> Do I understand correctly, that you say XDS will throw together for
> integration counts from many images even if they're spaced widely
> throughout the dataset, i.e. through the various passes?
>
> i.e. if I set up my data collection as 5 complete revolutions with
> low beam transmission - will XDS know to combine image 15 and
> 15+(360deg) and 15+(720deg) etc? By default? Or do I have tell it
> to do this explicitly, in which case, how?
>
>
> 2.
> If I have my 5x360 degrees of images, what metric / criterion do I
> use to decide whether to use only data up to 512deg or 839deg or
> 1469deg?
>
>
> Cheers
> Frank
>
>
>
>
>
> On 16/05/2013 18:03, Gerard Bricogne wrote:
> >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
--
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