Preparatory steps pyana version
The CS-PAD detector at LCLS is sensitive to temperature distortions and is not automatically dark-subtracted, unlike images from a synchrotron source. Because of this it is necessary to periodically collect a dark run with the shutters closed during an LCLS experiment. The dark run should contain on the order of at least 1000 images. Before an experimental run can be processed, this dark run must be averaged so that subsequent experimental data can have the dark subtracted from each image. Further, events during collection can necessitate a new dark, such as ice in the beam oversaturating creating dead pixels or a change in temperature.
In addition, averaging a lighted run in general is a useful tool for examining diffraction throughout a data collection run.
Finally, inactive and non-bonded pixels, as well as hypersensitive pixels and any beam stop (if used) should be masked out. This tutorial covers averaging dark and light runs, and creating mask images.
Create a dark average
To create the average, start by copying the dark.cfg file to your pyana directory:
$ cd ~/myrelease $ sit_setup $ cp /reg/d/ffb/cxi/temp/cctbx/tutorials/preprocessing/dark.cfg .
Open dark.cfg. It will look something like this:
# -*- Mode: Conf -*- # # Since mod_average must be run on a single host, it cannot be # submitted using lsf.sh. # # bsub -n 6 -J r0002 -o /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/dark_r0002.out -q psnehmpiq -R "span[hosts=1]" \ # "cxi.pyana -c ~/myrelease/dark.cfg /reg/d/ffb/cxi/temp/xtc/e236-r0002-s0*.xtc" # [pyana] modules = my_ana_pkg.mod_average num-cpu = 8 [my_ana_pkg.mod_average] calib_dir = /reg/d/ffb/cxi/temp/cctbx/cctbx_xfel/sources/cctbx_project/xfel/metrology/CSPad/run4/CxiDs1.0_Cspad.0 avg_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks stddev_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks max_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks detz_offset = 581 address = CxiDs1-0|Cspad-0 avg_basename = Ds1-r0002-avg stddev_basename = Ds1-r0002-stddev max_basename = Ds1-r0002-max
The config file is broken into three sections, the header, the pyana section and the modules section.
The comments at the top of the file include a bsub command. This is the command to be entered from the ~/myrelease directory to queue the job for processing. Bsub will submit the job, asking for 6 CPUs (-n), naming the job r0066 (-J), placing a log file in the -o location, and so forth. There are several queues available for use, the psnehmpiq is just one of them. The -R option restricts the job to running on a single host, which is required for averaging. Finally, the cxi.pyana command in quotes is the command that will actually be executed on the host computer, and specifies the config file and the streams to use. Note the asterisk, which allows averaging all the streams from a single run at one time.
Before executing the command, replace <username> in the command and in the config file with your user name.
Pyana operates by having a root section in the config file which specifies which modules to run. In this case we are running cctbx.xfel's mod_average module. If we were running multiple modules, we could chain them together here, and each module would be executed in sequence using the parameters specified below. We also request a number of cpus to parallelize the job with. Even though averaging must be done on a single host, on that host the processing can be split up among multiple cores. Note, other aspects of cctbx.xfel, such as indexing and integration, do not have this restriction.
The final section specifies parameters specific to averaging:
- calib_dir: this directory is provided by LCLS and includes the layout information of the 64 tiles of the CS-PAD detector
- avg_dirname: directory to place the average image
- stddev_dirname: directory to place the standard deviation image
- max_dirname: directory to place the maximum projection image
- detz_offset: experiment-specific number used to calculate the detector distance. This is the distance in millimeters between the sample interaction region and the far end of the detector stage. The XTC stream contains the distance from the detector stage to the detector. These two numbers are subtracted to calculate the distance between sample interaction region and the detector.
- address: the name of the detector in the XTC stream
- avg_basename: this name is prepended to a datestamp to name the average image
- stddev_basename: this name is prepended to a datestamp to name the standard deviation image
- max_basename: this name is prepended to a datestamp to name the maximum projection image image
Running and testing the command
Submit the job as specified above, using the bsub command in the header:
$ bsub -n 6 -J r0002 -o /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/dark_r0002.out -q psnehmpiq -R "span[hosts=1]" \ "cxi.pyana -c ~/myrelease/dark.cfg /reg/d/ffb/cxi/temp/xtc/e236-r0002-s0*.xtc"
Type bjobs to see if the job is running. When it is, follow the output thusly:
$ tail -f /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/dark_r0002.out
When the average has been completed, the output can be inspected using the "cctbx" image viewer:
$ cctbx.image_viewer /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks/Ds1-r0002-*.pickle
Averaging an experimental light run
Once the dark run is made, experimental runs can be averaged to see the extent of diffraction and potential pathologies in the run. Copy the average.cfg file from the tutorials directory to your pyana directory:
$ cd ~/myrelease $ cp /reg/d/ffb/cxi/temp/cctbx/tutorials/preprocessing/average.cfg .
The cfg will look like this:
# -*- Mode: Conf -*- # # Since mod_average must be run on a single host, it cannot be # submitted using lsf.sh. # # bsub -n 6 -J r0003 -o /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/average_r0003.out -q psnehmpiq -R "span[hosts=1]" \ # "cxi.pyana -c ~/myrelease/average.cfg /reg/d/ffb/cxi/temp/xtc/e236-r0003-s0*.xtc" # [pyana] modules = my_ana_pkg.mod_average num-cpu = 8 [my_ana_pkg.mod_average] calib_dir = /reg/d/ffb/cxi/temp/cctbx/cctbx_xfel/sources/cctbx_project/xfel/metrology/CSPad/run4/CxiDs1.0_Cspad.0 dark_path = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks/<name of dark average>.pickle dark_stddev = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/darks/<name of dark stddev>.pickle avg_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/averages stddev_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/averages max_dirname = /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/averages detz_offset = 581 address = CxiDs1-0|Cspad-0 avg_basename = Ds1-r0003-avg stddev_basename = Ds1-r0003-stddev max_basename = Ds1-r0003-max
The only difference is the addition of two parameters:
- dark_path: path to the average dark image created before
- dark_stddev: path to the standard deviation dark image created before
The job is ran as before using the bsub command in the header. It can be tested with cctbx.image_viewer in the same manner as well.
Information contained in the light average
Firstly, the light and dark images can be used to mask of any bad pixels, as detailed below. The light images (maximum in particular) also serve as a virtual, dark-subtracted, powder pattern that can used to inspect the data for diffraction. The light maximum should also flag up obvious errors in meteorology, because rings will be non-continuous if the quadrants are misaligned. Intensity values from the corners of the light average or maximum are also useful to estimate background when taking first guesses at thresholds for hit-finding and integration.
Creating a mask image
cctbx.xfel uses three images to create a mask. Those images, and the purposes of them are listed below:
- Average image from a dark run: pixels ≤ 0 are considered dead, pixels > 2000 are too sensitive
- Standard deviation from a dark run: pixels ≤ 0 are considered dead, pixels ≥ 10 are too uncertain
- Maximum projection from a lighted run (i.e. an experimental run): pixels < 300 are considered non-bonded or in a shadow. The presence of diffracting data is not needed, but also will not interfere.
To create the mask, execute these commands:
$ cd /reg/d/ffb/cxi/temp/cctbx/tutorials/scratch/<username>/ $ mkdir masks $ cd masks $ cxi.make_mask -m 10 ../darks/<name of dark average>.pickle ../darks/<name of dark standard deviation>.pickle \ ../averages/<name of light maximum projection>.pickle
A new image is created where each pixel has one of two values: -2 (masked out pixels), and 0 (good pixels). Note, the maximum projection minimum pixel value is overridden here to a value that is more sensible for the tutorial's lysozyme data. You will likely need to adjust this parameter based on your data (see below for details and for further overrides).
Use the resultant mask image during processing with cctbx.xfel by adding this flag to your pyana config file:
mask_path = <path to mask file>
In addition, a polygon and a circle can be specified using the --poly_mask and --circle_mask parameters:
$ cxi.make_mask --poly_mask=17,854,76,967,760,982,762,879 --circle_mask=855,939,103 Cspad-avg.pickle Cspad-stddev.pickle Cspad-max.pickle
This example creates a mask image with the polygon with points (17,854), (76,967), (760,982), and (762,879) masked out, and the circle with center (855,939) and radius 103 masked out. Only one polygon and circle can be specified at the moment.
The mask can be examined with cctbx.image_viewer.
Some additional options are available for specifying the details of the masking operation:
- -a, --avg_max (default 2000)
- specifies the maximum value in ADU a pixel in the average image can have before it's masked out. The minimum is always zero.
- -s, --stddev_max (default 10)
- specifies the maximum value in ADU a pixel in the standard deviation image can have before it's masked out. The minimum is always zero.
- -m, --maxproj_min (default 300)
- specifies the minimum value in ADU a pixel in the maximum projection image can have before it's masked out. Of the three parameters for controlling cutoffs in cxi.make_mask, the -m option is unique from the -a and -s options in that it will likely vary the according to your sample's background. Carefully examine the corners of your lighted maximum projection and choose a value lower than the ADU values displayed. Also note that there is no cutoff on the high end of the maximum projection image specified in the mask, as that is defined as the saturation value for the detector.
- -x, --mask_pix_val (default -2)
- specifies the value to use when masking out a pixel. In other words, bad pixels in images in the XTC stream will be replaced with this value.