Collect Tab

Overview

The Collect Tab is used for collecting test images and complete monochromatic, SAD and MAD data sets. Multiple run windows can be set up by creating additional Run Tabs.

Data collection runs

  • The Run tab 0 is the only run displayed when the collect tab is first opened. It is a special run, dedicated for taking single test images in order to test crystals and plan strategies for data collection. Only one image at one time can be collected from this run, so it is not possible to enter an end frame, although the drop down menu arrow next to the phi value can be used to quickly set up image collection at 90 degrees away from the first image. A resolution predictor widget displaying the resolution limits for the selected values of the detector, beamstop distance, and wavelength is available for this tab.
  • To create additional data collection run, click on the '*' tab below the '0' tab. Runs numbered 1 and above can be used to collect complete data sets at multiple energies. Note: When you create a new "run", the contents of the old run are automatically copied to the new run.
  • There is a limit of 17 runs that can be defined. Once this limit is reached, you must delete old runs in order to define new ones. Data already collected in not affected by erasing the run.

Data collection parameters and commands

Click on the links above for information on each parameter or input window.

  • If you input data collection parameters by hand make sure that you do not exceed the particular motor range. You can avoid this by selecting values within the range defined by the drop-down menu next to the input box. If you select a value outside this range, the name of the parameter will be highlighted in red. You will not be able to initiate the data collection.
  • A motor or parameter value displayed in red indicates that the motor is in a different position. The motor will move to the position in red upon starting data collection.
  • If you click on Default, the current values of distance, axis and energy will be selected and displayed. In addition, prefix will be set to 'xtal', directory to '/data/username'. The default detector mode, delta phi and exposure time are different for each beamline.
  • Click on Update if you wish to use the current motor positions for the data collection. This is useful if you have already set up the correct detector or beamstop distance from the Hutch Tab. If you have mounted a sample using the screening tab, update will also change the directory and image prefix names to the ones defined in the screening tab. Similarly, if you have done a MAD scan in the Scan Tab, the optimal energies for a MAD experiment will be copied to the run.

    • Important: Do NOT click on the Update button once you have finished editing the collection run. This will cancel all your edits.

  • Use the Delete button to delete the run that you are in. You cannot delete Run 0. Be careful when using this command. Once a run is deleted, you cannot return to it.
  • Use the Reset button to re-use a run. If you do this you should change the image name or the destination directory. If you do not change the names, Blu-Ice will create a subdirectory called OVERWRITTEN_FILES where the old files will be stored. This will protect the images from one accidental overwrite.
  • Another use of the Reset button is to edit data collection parameters after stopping a started run (for instance, to change the detector position). Note: if you click "Collect" immediately after editing the run, data collection will restart on the first image again. In order to continue collection at the current frame, double-click on the image name as explained in the Tips.


prefix
  • Filename root or file prefix
directory
  • Shows the directory in which the image will be saved. Only directory paths starting with "/data" are allowed. When you select this input box, a list of all the subdirectories in the current directory will be displayed as a drop-down menu. You can either select one of the existing directories, or type in a brand new one.
beam size
  • Beam size enables inputting various beam sizes for the data collection. Please see the Adjusting Beam Size section in the Sample_tab for more information.
detector
  • Specific values for the detector depends on the type of detectors. SSRL beamlines use so-called Pixel Array Detectors: Dectris Pilatus 6M, Eiger 16M and Eiger2 XE 16M.
  • The Pixel Array Detectors support shutterless data collection in which the shutter does not close for image readout except for the last image in a run or a wedge (if the run is being divided in multiple wedges) or before collecting inverse beam images. This works because the readout time is much shorter than the time taken to expose each frame. See information below about pausing the run in shutterless collection mode.
distance
  • Distance between detector and sample in millimeters; the minimum distance is usually between 90 and 110 mm, depending on the beamline.
beamstop
  • Beamstop distance in millimeters.
attenuation/transmission
  • Beam attenuation or transmission. Use to avoid overloaded spots when collecting low or medium resolution data from strongly diffracting crystals.
axis
  • Phi is the only choice unless the beamline is equipped with a Kappa diffractometer.
  • When Kappa is not set to zero, you must collect using Omega.
  • The rotation range of Omega is limited.
  • On beamlines with a Kappa diffractometer Omega and Kappa are usually locked to prevent hardware collisions, but can be unlocked if the experiment requires it: contact your support staff.
delta
  • Oscillation length per image. For the Pilatus and Eiger detectors a small delta (0.1 or 0.2 degrees) is recommended because it improves the data statistics and the detector readout time does not add up anything to the total collection time.
time
  • Length of exposure time in seconds. Note that if you change the oscillation angle per image, the exposure time should be changed by the same factor to obtain an equivalent exposure per image.
start
  • The number and phi value assigned to the first image.
end
  • The number and phi value assigned to the final image.
inverse beam
  • Rotates the crystal by 180 deg to collect the Friedel pairs for the input phi range.
  • When inverse beam is used for a MAD experiment, the inverse beam pass is collected before changing the energy.
wedge
  • The phi rotation range that is collected consecutively before rotating the crystal by 180 degrees (with inverse beam) or changing energy (during Multi-wavelength experiments).
energy
  • Energy or energies used for the experiment. As you enter an energy value, an empty box appears for further energy entries. The energy boxes can be auto-filled by clicking on the Update button after doing a MAD scan. If you wish to skip one or two of the selected energies, simple delete the value.
take microspec snapshot during run
  • This option is available when enabled from the Microspec Tab on beamlines equipped with an in situ microspectrophotometer. When checked, the software will automatically collect a UV spectrum after a number of frames entered by the user. The number of frames defaults to the amount specified by the wedge. The spectrum will always be collected at the same orientation given by the phi angle selected by the user (defaults to the data collection start). To calculate the optimal phi to monitor the spectrum, please refer to the documentation for the microspec tab.

Run Dose Estimate

  • The Run Dose Estimate window in the Collect tab provides information about X-ray absorption during data collection. The window displays experimental Dose Limits based on the type of experiment being performed, a calculated Predicted Dose based on crystal parameters and data collection Run parameters, and an estimate of the Exposed Dose as the experiment proceeds.
  • In the figure, the Run Dose Estimate window (highlighted in orange) shows the X-ray Dose Limit and displays a Predicted Dose based on the sample crystal and the Run parameters. This example displays a Predicted Dose estimate that exceeds the limit for data collection at room temperature.
  • The Dose Limit is defined as the dose which reduces the sample to half it's diffracting power or the loss of useful anomalous signal. The Dose Limit can be toggled by clicking on the Limit (blue indicates a toggle). Current options include:
    • Cryo (30 MGy) - Experiments performed at ~100 K
    • Cryo Se-MAD (5 MGy) - Seleno-met Multi-wavelength Anomalous Diffraction experiments at ~100 K
    • Cryo S-SAD (3 MGy) - Sulfur Single-wavelength Anomalous Diffraction experiments at ~100 K
    • Room Temp (0.38 MGy) - Room Temperature experiments
  • The Predicted Dose (the Average Diffraction-weighted Dose) is calculated using RADDOSE3D. RADDOSE3D is also a supported program that can be run independently from the command line.
  • The Exposed Dose is an estimate of how much dose the crystal has absorbed once data collection is running or has finished. The Predicted and Exposed Dose values will turn red to indicate a warning that they exceed the Dose Limit (see figure).
  • NOTE: The Predicted and Exposed Doses only apply to individual Runs since a translation of the sample to an unexposed area is assumed between Runs. If the crystal is exposed in the same position over multiple Runs, the total Predicted and Exposed Dose will be the sum of the doses for each Run.

Default Crystal Parameters

  • By default, a Predicted Dose will be calculated based on a number of Default assumptions:
    • The crystal is close to spherical or cubic in shape
    • The crystal is larger than the beam size
    • The protein is average in size
    • No heavy metals
    • Water solvent
  • WARNING: If the above assumptions do not adequately describe your crystal, the predicted dose will likely be underestimated which can lead to an over-exposure of your sample. In this case, use the X-ray Dose and the Crystal Size sub-tabs to enter the appropriate sample information (see below).

X-ray Dose Sub-Tab

  • Relevant information about the mounted sample will yield a more accurate Run Dose Estimate. Sample parameters can be modified in the X-ray Dose sub-tab (see figure).
  • Unit Cell, Monomers and Residues. The unit cell can be specified as well as the number of monomers in the unit cell and the number of residues in each monomer. Alternatively, the molecular weight of the monomer can be specified by clicking on the "Number of Residues" parameter (blue indicates a toggle).
  • Heavy Atoms. The type and number of heavy atoms in each monomer can be specified. For example, four iron and six selenium atoms in the monomer would be entered in the following manner: Fe 4 Se 6
  • Solvent Content. The percentage of solvent content can also be specified as well as the heavy atom concentrations in the solvent. The type and concentration (mmol/l) of each heavy atom type in the solvent can be specified. For example, concentrations of 100 mmol/l of P and 350 mmol/l of S and 50 mmol/l of Zn would be entered in the following manner:P 100 S 350 Zn 50. NOTE: Atoms lighter than oxygen should not be included.
  • Once the Dose Parameters in the X-ray Dose sub-tab have been entered, click on "Apply" to update the Predicted Dose Estimate. Values highlighted in red will be updated and a new Predicted Dose will be calculated. Use the Cancel button before clicking Apply to discard changes to the parameters.
  • NOTE: The X-ray Dose sub-tab parameters apply to the crystal that is currently mounted on the goniometer and therefore these parameters apply to all Run tabs.
  • At the bottom of the sub-tab, the crystal shape and size are displayed as specified in the Crystal Size sub-tab. When a sample is first mounted, the crystal shape and size default to a sphere with radius about twice the size of the beam in Blu-Ice. The Crystal Size sub-tab can be used to redefine the crystal shape and size for a more accurate estimate of the dose.

Crystal Size Sub-Tab

  • The Crystal Size sub-tab (figure below, left) provides an interactive tool to define the shape and size of the mounted sample to give a more accurate estimate of the absorbed dose. A yellow overlay that defines the shape and size of the crystal is displayed on the camera view of the sample.
  • When a sample is first mounted, the shape defaults to "Default Sphere(2.3 X beam size)". The default state assumes the crystal is approximately the same size or larger than twice the beam size in Blu-Ice. Thus, the diameter of the Default Sphere is set to 2.3 times the current beam size in Blu-Ice (i.e. the diameter is set to the Full Width of the Gaussian beam).
  • Additional shapes with handles are available in the drop down menu to better match the crystal shape and size for a more accurate Dose Estimate (figure below, middle). Choose the shape that most closely resembles the shape of the crystal. Resizing handles will appear on the yellow overlay for Sphere and Rod (figure below, right). If the beamline provides two camera views, either view can be used to adjust the size of the overlay. Grab and move the handles with the mouse (click and hold) to adjust the size of the circle/box to best fit the crystal. Handles that are colored the same adjust the shape in the same way. The crystal should be rotated about the Phi axis to verify the crystal shape and size are adequately defined.
  • NOTE: The Predicted Dose is immediately updated whenever the shape is changed or when the handles are used to change the size of the crystal.
  • When Cube or Rectangle are selected (figure below, left), the crystal should be rotated about the Phi axis to align one of the faces of the crystal approximately normal to the camera view. Once the face of the crystal is aligned, two handles will appear foradjusting the size of the yellow overlay to match the face of the crystal (figure below, middle). Use the +90 button to verify the yellow outline aligns with the crystal as it is rotated. For the Rectangle shape, an additional handle (green) will be available. Use the green handle to define the 3rd dimension of a rectangular crystal (figure below, right).
  • To use a different orientation of the crystal as the initial "Face" of the Cube or Rectangle, rotate Phi to the new location. The Cube or Rectangle overlay can now be resized at this new Phi position.
  • NOTE: If the crystal is not clearly visible, the crystal can be rastered using low-dose X-rays to provide a reasonable estimate of the crystal size. See the Rastering Tab for more information on how to raster the crystal.

Diffraction Quality Strip Chart

  • The strip chart at the bottom of the Collect Tab displays a few key data processing statistics in near real time (see figure). The program Interceptor is used to quickly process each image as it is produced and the results are displayed in the strip chart. Resolution, a Quality Score, the Number of Spots, and the Likelihood of Spot Splitting are determined for each image. The Score is primarily based on resolution, with penalties and bonuses applied for number of ice rings, max intensity between 15-4.5Å, and spot elongation. Thresholds can be set to indicate the number of images/frames that meet a particular requirement (e.g. minimum resolution of 3.0 Angstrom) (in color) and those that do not (in black). The chart can be paused by checking the Pause checkbox. The Score, Spots, and Splitcharts can be toggled on or off by selecting the respective checkbox. The window can be resized vertically by clicking on and dragging the square in the upper right corner of the window.
  • WARNING: These on-the-fly calculated statistics are not as accurate as fully processed data. For a more accurate assessment of your sample, please check the Automated Processing directories for your particular dataset.
  • Hovering over the chart area will produce a cross cursor which can be used to select an area to zoom into (click and drag). Multiple zooms can be used to see individual data points when collecting images rapidly. Use Zoom Out in the context menu (right click on the chart area) to zoom out to the previous zoom area. Select View All to see all the data on the chart. Hovering over a data point will bring up its value and image number. Clicking on a data point will display the associated diffraction image in the Diffraction Sub-Tab. To resume displaying images as data are collected, uncheck the Hold Image checkbox in the Diffraction Tab.

Automated Data Processing

  • Once a run has completed with 5 or more images, the data are automatically processed providing quick feedback on the quality of the dataset. If a spreadsheet has been assigned to the cassette, the processing run information (and selected results) will be written into the Sample database. Once in the Sample database, the processing information will be displayed in the new Processing Tab. A complete description of automated processing and the new Processing Tab can be found here.

Helical Data Collection

  • The Helical Path Sub-Tab is located next to the Crystal Size Tab and allows collection of oscillation data while the sample is translated along a predefined path. On BL12-1 and BL12-2, the software collects oscillation data in a continuous shutterless mode by setting the speed of the translation for a constant exposure time. On BL14-1 and BL9-2, the software collects an oscillation image then translates the sample to a new position and collects the next image, etc.
  • The best strategy to optimize diffraction resolution while minimizing the effects of radiation damage is to expose the largest possible volume of your crystal. When the crystal size is larger than the largest beam size, use the Helical Data Collection option to translate the entire crystal through the beam. First maximize the vertical beam size to match the vertical size of the sample (if possible) and then define a horizontal path to translate the crystal.
  • The Path is defined by creating a graphic overlay on the live video stream of the sample in the new Helical Path Sub-Tab ( described in detail below).
  • Helical data collection is initiated and managed in the same way as standard Rotation data collection which is described extensively above in the Collect Tab Manual.
  • Any number of Frames can be specified. For BL12-1 and BL12-2 where shutterless data collection is employed, the speed is adjusted for an even exposure of the sample as it is translated and rotation images are collected.
  • The Default Button will set a default number of Frames for the existing Path so that the exposure overlap is ~1/2 the horizontal Gaussian beam size. If a larger dataset is desired, the number of frames can be increased with a commensurate increase in the overlap of the exposure area (typical strategy for collecting a full data set from a single crystal). Likewise, exposures can be set farther apart at the expense of collecting fewer Frames.

Helical Path Interface

  • The Helical Path interface provides a graphical overlay on the live camera view of the sample to set a path for helical data collection (see figure). One Path can be defined for each Run and up to 16 Runs (and thus 16 Paths) can be specified for each mounted crystal (or multiple crystals on a mesh). Choose one end of your sample for the starting point of the helical Path. Align the starting point in the center of the beam (at phi and phi +90). Press "Save Path Start". Choose the end of the helical path and center it at the beam center. Press "Save Path End".
  • NOTE: it is critical to rotate Phi 360 degrees to verify the positioning of the Path is correct on the sample.
  • NOTE: in crystal overlay displayed, the vertical beam may appear to change in size because this image is a projection at different Phi rotation values.
  • To modify the Path, press "Define New Path".
  • By default, only the associated Path is shown for the selected Run as an overlay on the sample camera video. All Paths can be shown by clicking on the "Show All Paths" checkbox. In addition, results from the Raster Tab can be overlaid on the sample by selecting the Show Raster Tab Results checkbox.

Starting a Data Collection Run

  • After setting all parameters to your desired values click Start to collect an image.
  • The network status of your Blu-Ice Client must be Active to collect an image.
  • If you have created a few runs and start data collection from a previous "run", Blu-Ice will collect frames from the current run and the following runs (Note: Blu-Ice will not recollect frames from an already completed "run"). This allows users to collect multiple data sets (eg., low resolution pass, high resolution pass or different wavelengths) using different "run" windows.

Run Time

  • Run Time shows the time remaining for the data collection run. If new values of data collection parameters are entered in the run definition, the run time is recalculated. During the run, the run time is updated after every image.

Data File Naming

  • Each data file in your selected run sequence is named in the following way: 'file prefix'_'run number'_'energy number'_'image number'.img.
  • For example, a file could be named data_2_E2_010.img. This image is in the 10th frame collected in Run 2 at the 2nd input energy.
  • With only one energy level selected, the file would be name 'file prefix'_'run number'_'image number'.img.

Run Sequence

  • The run sequence depends on the values for phi, wedge, energy or inverse beam that you choose. The image collection order will be displayed in the 'run sequence' window. Below are examples of some possible run sequences.
  • Note: Once the current run is finished, the software will go on to collect any unfinished runs (paused or inactive) following the current one.

    One Energy, Inverse Beam Off,
    Phi < Wedge Size
    (simplest case)
    example:
    test_1_001
    test_1_002 
    test_1_003
    (phi: 0-3 deg, wedge: 3 deg, 1 energy, inverse beam off)
    Two or more Energies, Inverse Beam Off, Phi < Wedge Size

    example:
    test_1_E1_001
    test_1_E1_002
    test_1_E1_003
    test_1_E2_001
    test_1_E2_002
    test_1_E2_003
    (phi: 0-3 deg, wedge: 3 deg, 2 energies, inverse beam off)
    Two or more Energies, Inverse Beam On, Phi < Wedge Size

    example:
    test_1_E1_001
    test_1_E1_002
    test_1_E1_003
    test_1_E1_181
    test_1_E1_182
    test_1_E1_183
    test_1_E2_001
    test_1_E2_002
    test_1_E2_003
    test_1_E2_181
    test_1_E2_182
    test_1_E2_183
    (phi: 0-3 deg, wedge: 3 deg, 2 energies, inverse beam on)
    Two or more Energies, Inverse Beam On, Phi > Wedge Size

    example:
    test_1_E1_001
    test_1_E1_002
    test_1_E1_181
    test_1_E1_182
    test_1_E2_001
    test_1_E2_002
    test_1_E2_181
    test_1_E2_182
    test_1_E1_003
    test_1_E1_183
    test_1_E2_003
    test_1_E2_183
    (phi: 0 - 3 deg, wedge: 2 deg, 2 energies, inverse beam on)

Image display commands

New images are displayed to the left of the collect tab as soon as they are written to the users' area. Older images can be displayed and inspected by launching the program ADXV.

brightness
  • The default image Brightness value is set to 400. Change this value for a desirable contrast between spots and background.
zoom
  • Click on this to zoom in or out of the diffraction image.
move arrows
  • Pans the image within the display box.
Alternative image viewers
  • Allows the user to inspect and analyze diffraction images with ADXV.
Select image
  • Allows the user to select the previous and next images in the run sequence with the + and - buttons. To use this tool while data collection is ongoing, click the Hold image check box.

Useful Tips

  • Subdirectories are automatically created.
  • The next image to be collected is colored red in the 'Run Sequence' box. You can jump to a different image if you double click on the desired image name.
  • Pressing the "Pause" button stops the data collection after completing the current frame, or, in the case of shutterless data collection, after completion of the current wedge.
  • Pressing the "Abort" button immediately stops the data collection.
  • Double-clicking on an image automatically starts ADXV.