Screening Tab

• Overview
• Uploading Crystal Information in a Spreadsheet
• Viewing and Selecting Samples
• Customizing the Spreadsheet Layout
• Port Status Indicators
• Screening Configuration
• Screening Action Sequence
• Starting and Stopping Screening
• Screening Status
• Screening Task Window
• Sample and Hutch Real-Time Video Display
• Data Collection Strategy
• Collecting Data after Automated Screening
• Saving Screening Results

Overview

The Screening Tab (Fig 1.) provides an interface for automatically screening samples using the SAM system. With this interface, the user selects multiple samples of interest from an embedded spreadsheet and defines the actions to be performed on each sample. Once started, the interface can run with minimal supervision until all of the samples have been screened identically.

[Fig 1] The Screening Tab in Blu-Ice

The screening tab is divided in four different parts (modules or widgets) by one vertical and two horizontal lines. The widgets can be resized by clicking on the resize button on the dividing line and dragging it in the desired direction.

Before automatically screening samples in a cassette, the system must first have some basic information about each sample in the cassette. This information is entered into an Excel spreadsheet and then uploaded through a web browser. Templates for the spreadsheet can be downloaded from the Sample Database. The template can be edited using Microsoft Excel or the spreadsheet editor which comes with OpenOffice running on a Unix system. Detailed instructions for the content of the spreadsheet can be found in the Users' Guide. Once the information has been entered into the spreadsheet, the spreadsheet is then loaded into Blu-Ice in one of 2 ways:

1. Via the SSRL database which is accessed by clicking the Web button and choosing the Spreadsheet option on the Cassette Definition Import Menu (Fig. 2) on the Screening tab. Please see the Users' Guide for detailed instructions on how to use the web interface for loading Excel information into the database and assigning the information to a particular cassette position at a particular beamline.



[Fig 2] Cassette Definition Import Menu

2. By direct upload from the local file system by first selecting the left, middle or right cassette position in the Cassette Definition Import Menu (Fig. 2) and then clicking the Import button; for this option, the Excel file must be first transferred to a local SSRL computer at the beamline or processing computer. Verify that the cassette position is correct.

Once the information has been loaded correctly, using either method, the spreadsheet information should be displayed in a table format (Fig. 3).

[Fig 3] Spreadsheet display window

Viewing and Selecting Samples

The Spreadsheet window (Fig. 3) can be used to select the samples to be screened. This window will list the samples in the order that they were defined in the original Excel spreadsheet.

Only three of the columns present in the spreadsheet are used by the screening system as described here:

• Port: refers to a port in the cassette. The robot will go this port to retrieve the sample.
• CrystalID: this label is used by the screening system to generate filenames. It is recommended that a unique ID be used for each sample.
• Directory: If provided, this field will be used to generate subdirectories for each sample under the root screening directory. The default template Excel spreadsheet uses the Port as the subdirectory name. However, if no spreadsheet is loaded into Blu-Ice, all the images will be written to the same directory.

Other columns are displayed for information purposes. They can be excluded or reordered as explained below.

To the left of the Port column are check boxes used to select samples for screening (the box is blue when selected). These individual check boxes can be clicked to select or deselect all the samples. At the top of this column is a Global selection check box that can be used to select or deselect all the samples. Please note: By default, all samples are selected when the spreadsheet is first loaded into Blu-Ice.

Port Status Indicators

The leftmost column in the Sample Information Widget (Fig. 3) indicates the status of the cassette port.

• The indicator shows the sample that is currently mounted on the goniometer.
• The indicator shows which sample will be mounted after the screening actions are completed for the currently mounted sample. The next sample scheduled for mounting can be changed interactively by clicking on the Port status field of another sample.

Customizing the Spreadsheet Layout

After successfully importing the cassette and sample information into Blu-Ice, the spreadsheet is displayed in a table format. The number and format of the spreadsheets displayed can be customized by the user by using the following tools:

1. The View pull-down menu to the left of the Column headers (see Fig. 5).
2. The column editing menu displayed when moving the mouse over the column headers (Fig. 6a).
3. The mouse "click and drag" commands to change the size and positions of the column (Fig. 6b and c)

The spreadsheet View pull-down menu (see Fig. 5) offers the following options: Full View: Displays all the columns that are present in the original Excel file. Simple View: Shows only the Port, CrystalID, and Protein columns. Results View: Shows the results returned from WebIce, including the Score and UnitCell for each sample. Save Custom View: Saves the current view for use with future Blu-Ice sessions. The format information file is saved to the directory /home/userid/.bluice/show_column. If the user saves the custom column outlay as ".dflt.cln", it will become the default for subsequent sessions. Load Custom View: Loads a previously saved view. Refresh: Reloads the spreadsheet contents.

[Fig 5] File View Menu

[Fig 6a] [Fig 6b] [Fig 6c] Selection and configuration of spreadsheet columns

The column editing options (available by placing the mouse over the spreadsheet field header) are: Hide (Fig. 6a): Clicking on this drop-down menu option hides the column from the current view. Insert (Fig. 6a): Clicking on this option displays a list of spreadsheet columns. The user can select one of them to be inserted to the left of the current column. Note: This option is available only for hidden columns; displayed columns will be grayed out in the selection list. Maximize(< -max- > in Fig. 6a): This expands the width of the column to match the maximum width of its contents. Resize (Fig. 6b): The column can be resized by moving the mouse to the right of the column header until a tab symbol appears; click and drag the mouse toward the right to increase the column width; or toward the left to decrease it. The column header is highlighted in green while this command is active. Move (Fig. 6c): Clicking on the column header allows moving the column to a different position, by dragging the mouse either to the left or right. The column header is highlighted in green while this command is active.

Screening Configuration

Many of the data collection parameters used during screening can be set using the interface on the screening tab shown in Fig. 7.

The Directory entry box specifies where the screening results will be stored. The user's Unix account must have write permissions to this directory, otherwise screening will not start. The Default button changes the directory to the current user's top level data directory. Subdirectories can be added. During screening, the directory will be automatically created if it does not exist. If the sample information contains a directory for each sample, the directories will be subdirectories of this entry box.

The detector mode can be selected, as explained in the Collect Tab documentation

The detector distance and beamstop distance can also be specified. The detector and beamstop will move to the requested distances when images are collected during screening. Clicking on the Update button will change the values in the entry widgets to match the actual physical values of the detector distance and beamstop distance. Clicking on the Update button will not move the detector or beamstop from their current positions.

Attenuation can also be selected to move filters into place during screening to attenuate the beam. Clicking on the Update button will change the value in the entry widget to match the current value of Attenuation. Update will not move any filters into or out of the beam path.

[Fig 7] Directory and Detector Interface

Screening Action Sequence

The Screening Sequence interface (Fig. 8) allows the user to configure actions to be performed for each sample. The interface consists of a list of actions with a check box next to each action. An action with a selected checkbox indicates that it will be performed during the screening process. Some selections are permanent and will be performed automatically for each sample during screening. These permanently selected actions are "Mount Next Crystal", and the first "Collect Image". The dark green color of these check boxes indicates that they cannot be deselected. "Loop alignment" can be deselected, but if this is done the following "Stop" will become automatically selected; this is done to ensure that the sample is centered, either automatically or by the user, before diffraction shots are collected.

[Fig 8] Screening Action Interface

The following is a summary of the available screening actions:

• Mount Next Crystal - Mounts the sample indicated by the indicator in the Sample Information Widget. If a sample is already on the goniometer, it will be dismounted and returned to the port from which it came. This action also creates a directory for the output files to be generated for the new sample. Once these actions are complete, the indicator will be replaced by the indicator.
• Loop Alignment - This action performs an automated loop alignment procedure. Video images of the sample are taken at different magnifications and phi rotation angles to determine the position and face of the loop. The loop is then centered on the beam. Note: If this check box is deselected, the next Stop will be automatically selected, to provide a chance for the user to center the sample manually.
• Raster Alignment - This action performs an automated crystal alignment procedure using low-dose X-rays. The Raster Parameter Window appears by hovering over Raster Alignment (Fig. 9A & 9B ). A choice of beam size can be selected for the rastering process (a larger beamsize is faster; a smaller size provides more precise alignment). On beamline 9-2, only 2 choices are provided; one for crystals smaller than 200 um and for crystals larger than 200 um. If you have a mix of crystal sizes, use the <200 um option. The default Transmission and Exposure Time Per Grid Point is displayed for the beam size selected. If a different value is used, a warning indicating a value other than the default value is being used. The "Reset to Default" button allows the default parameters to be restored. The size of the raster is determined by the Loop Alignment procedure (when Raster Alignment is selected, "Loop Alignment" is automatically selected). If rastering fails, the sample will be aligned to the center of the loop.

  On BL12-2 an estimate of the crystal size is determined from the rastering results which can be used in subsequent Collect Image actions (i.e. for collecting snapshots or collecting full data sets). Otherwise the beamsize specified in the screening configuration (Fig. 7) will be used for subsequent actions.



[Fig 9] Rastering options A: example default settings, B: when the X-ray dose per grid point is increased

• Stop - is optional for pausing the screening process (unless Loop alignment is deselected) allowing manual centering of the sample. Normally this is not required if the loops and crystals are smaller than the beam size. To continue the screening process, depress "Start" on the Screening Action interface (Fig. 8).
• Collect Image - This action performs data collection of single images or it can be used to collect complete data sets. The Collect Image parameter window appears by hovering over Collect Image (Fig 10).
• Setup for Dataset button - Clicking on this button activates a single Collect Image run with default values for Delta, Time, and # of images/Phi Range.
• Match Beam Size check box - uses the estimated crystal size from rastering to set the optimal beam size, otherwise it uses the beam Size selected in the Screening Window.
• Delta - angular range of Phi (degrees) per image.
• Time - exposure time (seconds) per image.
• # of images - can be used to collect a series of images (starting from the current phi angle) for use in cell refinement in MOSFLM for example or to collect a complete dataset. It is highly recommended to center the crystal using rastering when collecting data sets.
• Rotate by - This field is available in the 2nd and 3rd Collect Image actions and indicates the Phi angle (degrees) relative to the first Collect Image. The default values are 90 and -45 degrees for the 2nd and 3rd Collect Images, respectively.

[Fig 10] Collect Image

The image filename is constructed as ID_image# with extension .img for Q315 images, extension .mccd for MAR325 images and extension .cbf for Pilatus and Eiger images. The video image file is in JPG format.

Starting and Stopping Screening

Once the samples have been selected for screening and the screening actions have been configured, the screening process can be started. The Screening Control Interface (Fig. 11) is used to control the screening process.

[Fig 11] Screening Control Interface

A summary of each button in the Screening Control Interface is described below:

• Start - initiates the screening process. The commands that are selected in the action sequence menu will be executed sequentially and will loop until a stop is encountered or until there are no longer any 'selected' samples.
• Stop - stops the screening process after the current action completes.
• Dismount - dismounts a sample from the goniometer and returns it to the cassette.
• Reset Parameters - replaces the all screening parameters (including Raster Alignment) with default values.

Screening Status

The screening status is displayed just above the Screening Action Interface.

The left most column in the Screening Action Interface is used to indicate the starting point of screening or the current action. The arrow beside an action will indicate that the screening sequence will start with this action. Once the action sequence is started, a indicator will point to the action currently being carried out. For example, when the robot is in the process of dismounting one crystal and mounting the next, the window will display the following:

during loop alignment the following will be displayed:

and during crystal rotation:

When screening is stopped, the arrow can be moved by clicking in the left column next to a different action. This is useful for repeating or skipping an action.

Additional status indicators can be found to the right of the Screening Task Window (Fig. 12). These indicators are described below:

• Current: shows which crystal is currently mounted.
• Next: shows which crystal will be mounted next.
• Mode: indicates whether the robot is in automatic or manual mode.
• Synched: indicates whether the robot is synchronized with the spreadsheet.
• Robot: indicates one of three states of the robot:
• The robot is in a normal state, ready for operation.
• The robot is currently performing an action.
• The robot requires user support staff inspection.

[Fig 12] Screening Task Window

Screening Task Window

The Screening Task Window (Fig. 12) displays a list of all the samples and actions to be performed based on the crystals and actions selected. Entries displayed in black in this task window will be executed when "Start" is clicked. (If there are no "Stop" actions for example, the entire list will be black and will be carried out to completion.)

The top action in the list will be highlighted in purple if it is currently being executed, and the action will be removed from the list when completed. If there is a "Stop" selected in the Screening Action Interface, the actions up to that point will be black and the remaining actions will be grayed out.

Sample and Hutch Real-Time Video Display

The image of the sample can be viewed in this window by clicking on the Sample Tab. This is the default video display during sample mounting, loop alignment, crystal rotation and image collection.

The sample can be manually centered by 'pointing and clicking' on the part of the sample to center. This behaves exactly the same as the Sample Window in the Hutch Tab. The zoom level can be selected and phi can be rotated from this window.

The Video Snapshot button can be used to save an image of your crystal in jpg format. You will be prompted to enter an image file name.

Selecting Hutch or Robot will display video feeds of the diffractometer, sample surroundings and robot. See the Video Widget documentation for a description of all the video presets.

Data Collection Strategy

[Fig 13] Link to WebIce

Choosing the Web -> Webice option (Fig. 13) will open a browser window and take you to the Screening tab of WebIce. If you have selected "Stop" after collecting two images in the Screening Action Interface (Fig. 8), then the View Strategy button in WebIce will be active, and you will be able to view the results of strategy calculations carried out in mosflm. See the WebIce guide for more information. The Autoindex tab in WebIce can be used to calculate a strategy for any samples for which you have collected two test images using the screening interface.

Collecting Data after Automated Screening

If your sample isn't already mounted on the goniometer, there are two methods for mounting crystals from a cassette and proceeding with data collection:

Option 1

• Use the Sample Mounting Robot Control Window in the Sample Tab to mount and dismount individual samples.

Option 2

• Deselect all the samples in the Sample Information Widget. Then select a sample to mount.
• In the Screening Action interface, select the "Stop" which follows "Loop Alignment".

Once the sample is mounted as described above it is possible to refine the crystal centering; try to improve the diffraction by sample annealing or washing ice off the sample; collect MAD or excitation scans; or collect a data set.

Saving Screening Results

All the information entered into the screening spreadsheet is saved in a database. You may save a spreadsheet to the local file system using the Export button above the spreadsheet display window (Fig. 13). Alternately, you may access the Sample Database via the crystal server. After logging in, the browser displays a list of the spreadsheets previously uploaded to SSRL together with a link to "Download Results" for each spreadsheet. If a spreadsheet has been used during automatic screening, the image analysis and autoindexing results will be in the spreadsheet.