To begin, set up the legend on interface, then navigate to application and click run. In the run application window, choose the Ptolemy application. Set main to the Legendon computer in scope and camera to the respective computers.
Now import presets in the preset manager. Within the square targeting node settings, ensure that both sort targets by shortest path and enable auto targeting are selected. In the square node settings, confirm that the wait for node to process the image is checked.
Add the square preset to the list if it's not already included. Under advanced settings, check set these apertures while imaging and ensure the aperture values are correct. To adjust whole targeting node settings, check allow for user verification of selected targets.
Uncheck both queue up targets and skip automated hole finder. In the whole node setting, check wait for a node to process the image and the whole preset is in the list on the right. In advanced settings, check set these apertures while imaging and confirm the aperture values.
In the exposure targeting node settings, select allow for user verification of selected targets. Ensure the Q up targets and skip automated hole finder are unchecked. To configure the exposure node settings, ensure the wait for a node to process the image is unchecked.
Confirm that the exposure preset is included in the list on the right. In advanced settings, select set these apertures while imaging and verify that these aperture values are accurate. In the focus node settings, ensure wait for a node to process the image is unchecked.
Verify that the auto focusing preset is listed on the right and set the desired autofocus accuracy to four times 10 to the negative six meters. In the focus node focus sequence, activate only two beam tilt auto focusing steps. To adjust the Z focus node settings ensure that wait for a node to process the image is unchecked.
Confirm the whole preset is listed on the right and set the desired auto-focus accuracy to five times 10 to the negative five meters. In the Z focus node focus sequence, enable only two low magnification stage tilt steps. After determining the grid Z height and collecting an atlas, use the square targeting node in Ptolemy to locate squares.
Observe the blue circles on each square. Hover over each blob to view their size as computed by Ptolemy and record the sizes of the largest and smallest blobs. To modify the threshold settings, adjust the minimum and maximum filter range to include desirable squares and exclude undesirable ones.
Press the fine squares button on the top toolbar. Fine tune the filter range until the fine squares feature effectively targets the squares. In the acquisition settings, select appropriate values for the maximum number of targets and number of target group to sample.
Once the parameters are satisfactory, click the play button to proceed. Then utilize the ruler tool in the whole targeting node to measure the diameter of a hole. In the template settings, ensure the measured diameter into final template diameter and click test.
Adjust the diameter value until all holes display bright white peaks at their centers. Click test in the threshold settings. Modify the A value until the binarized image distinctly shows white areas solely where holes are present.
In the blobs settings, measure a minimum distance from the edge with the ruler and input that value to exclude border targets. Filter blobs based on size, roundness and desired quantity. Hover over blobs to view their values and click test to review the current settings.
Enter the radius of holes and the spacing between them in the lattice settings. Then click the 42 button to gauge the reference intensity value of a vacuum area. In the acquisition settings, check use subset of the acquisition targets and set the sample maximal value to a low number such as two.
Define a broad range of ice thickness means in standard deviations. Use test targeting to randomize target selection based on the above values. Click the play button to proceed once all settings are satisfactory.
In the whole settings, set the shell script to the hlfinding. sh script path in the Ptolemy installation. Set the minimum score to less than zero.
Input the radius of the holes then click the show value button to measure the reference intensity value of a vacuum area. Click test to find the lattice of holes. In acquisition settings, check use subset of the acquisition targets and set the sample maximal value to a small number such as four.
Set a wide range of ice thickness means and standard deviations. Click the play button when satisfied with all settings. Legendon will perform centric focus and collect high magnification images which can be seen in the exposure node.
Check the next exposure targeting image to see if the settings are sufficient. Once satisfied, uncheck allow for user verification of selected targets in the exposure targeting and whole targeting settings. Once the grid is done screening, click file followed by exit to close Legendon.
To set up smart Legendon auto screen, execute smart Legendon's autoscreen. py in a terminal window. Select GY and input a comma-separated list of grid slots to the screen.
Then input full for the workflow. Input the template session name for new sessions and the template sessions Z height value. A GY will open to input the session name and select project associations for each grid.
Leave the microscope unattended or monitor it in Legendon, Appian and the microscope computer.
