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Frank, Kay or Wolfgang can obviously answer better, but I am pretty sure the answer to your second question is "no". XDS does not consider images collected at 0 and 360 to be "one image". In fact, I think the "phi direction" is internally represented as "detector Z", where "Z" has units of the ordinal "frame" number in the file name, except that "Z" does not have to be an integer. At the scaling or "CORRECT" step, these independent observations at different "detector XYZ" are combined together as "equivalent observations", but before that they are background-subtracted and integrated separately. It is an interesting question, I think, where, exactly, XDS starts to fall apart as you divide the photons over more and more images. You can, however, make the images "one" by physically adding them together with fit2d: http://www.esrf.eu/computing/scientific/FIT2D/ which I think can work with Pilatus-style CBF images. Then you can take your 5x360 images and make new "datasets" from them and see how XDS or other integration/scaling packages perform when the same photons are divided over more or fewer images. -James Holton MAD Scientist On 5/16/2013 10:25 AM, 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