Setting up the desktop and microscope

1. Log into computer (ask Dr. Wheeler for the login information).
2. Right click on past folders found on the desktop and select ‘delete’.
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Ensure that the past folders are uploaded to both the portable external hard drive and the Wheeler Lab OneDrive before deleting from desktop. The disk space on the desktop is limited compared to the number of videos we take, so it needs to be cleared at the beginning of filming to ensure there is enough space on the disk for the new videos.
3. Open up IC Capture 2.5 found on the taskbar at the bottom of the screen.
4. Plug in the black microscope camera cord into one of the USB ports. This will allow the camera to be selected as a device on IC Capture 2.5. Remove the blue microscope cover and set aside.
5. Select DMK 37BUX178 and click ‘Ok’ to open up the camera in the computer program. A box containing the live camera footage will appear on the screen and should be dark.
6. Select ‘Device’, select ‘Properties’, and match the settings below to the Device Properties on the screen:
1. Brightness = 200
2. Gain = 0.00 dB
3. Exposure = 1/4608 s
4. Auto Reference = 128
5. Auto Max Value = select ‘Auto’
6. IMX178 LLP Gain and Highlight Reduction are unselected.
7. Select ‘Ok’ in Device Properties to save the settings.
8. Turn on transmitted light on the Zeiss Stemi 508 by pushing in the lower knob found on the right side of the stand.
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Ensure that the light intensity is set to the max by turning the bottom knob on the stand all the way from you until the light stops increasing in brightness (the knob does not have a stopping point when its at its max).
9. Move the black switch on the back side of the camera port from right to left to open up the lens (this diverts light from one ocular to the camera).
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Zeiss Stemi 508 Microscope manual:

MULTILINGUAL_short-manual_Stemi-508.pdf
10. On the right side of the base, align the two yellow arrows with each other on the dial that controls the mirror.
11. Change the magnification on the Zoom knob to 2.0x for 96-well plates (1.0x or 0.8x for 24-well plates)
12. At the top of the screen, the video settings should match the settings below:
1. FPS = 16.00
2. View percentage = 30%
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The View Percentage can be increased, depending on how big you want the view of the microscope on the screen to be. When the View Percentage is increased, the tab in which the live feed is on will need to be expanded right and down to eliminate the scroll bars. The View Percentage does not change what is recorded, just what the user sees on the screen.
3. Video File Type = Y800 (2048x2048)
13. On the top of the screen is the Region of Interest settings, select the icon highlighted in blue below.

14. Match the Select Region of Interest settings to the dimensions below:
1. X1 = 0 X2 = 1616
2. Y1 = 0 Y2 = 1616
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Do not type the dimensions with a comma, as it will default back to the full dimensions. The ‘Width’ and ‘Height’ should autofill to the dimensions that were used above, but ensure that they are both 1616.
15. Save the Select Region of Interest by clicking the magnifying glass with a plus sign icon with the word ‘select’.

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The part of the whole camera frame should be reduced significantly to only film the well of interest.
16. Place an empty 96-well plate on the base of the microscope and make sure that it lines up perfectly to what you can see on the screen. Four sides of the well should be directly touching the edges of the frame on the screen and the whole well should be seen.
17. Adjust the course focus knob on the right side of the stand by turning it toward and away from you to fully focus the well to where the edges of the well are not blurry.
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This can be adjusted throughout the experiment when there are miracidia in the well to focus the microscope on.

Recording settings

1. Select ‘Capture’ and ‘Toggle Recording Info Dialog’
2. In the Codec section, select the yellow gear icon
3. The Recording Settings should match the settings below:
1. Container Type = AVI
2. Video Compressor = Uncompressed = Y800
4. Save the Codec settings by selecting ‘Ok’ if they were already set at these settings, or select ‘Apply’ if the settings needed to be switched to the correct settings.
5. In the Filename section, select the film strip icon.
6. Check the box to ‘Enable automatic video filename generation’.
7. Select ‘Change…’ to change the Target directory:
1. Select ‘Make New Folder’
2. Make a new folder under the Desktop directory with the date typed as yearmonthday (i.e., 20230801)
3. Select ‘OK’ to save
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Double check that the path of the Target directory is correct.
8. Filename prefix should be labeled as p01_t00_ for the first video of the day.
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The Filename prefix will change based on how many plates and time points are being recorded that day.
9. Check the box ‘Index.’ The Index should be at 1 when initially recording videos.
10. Save the Video File settings by selecting ‘Ok’ if they were already set at these settings, or select ‘Apply’ if the settings needed to be switched to the correct settings.
11. In the Information section, the Limit should be 00:00:15. With the rest of the settings at 0.
12. If this is not the case, select the clock icon to change the recording time:
1. Check the box ‘Stop recording after’.
2. Type in 00:00:15 under ‘Auto-Stop by Elapsed Time’.
3. The rest of the boxes should remain unchecked.
13. Save the advanced settings by selecting ‘ok’ if they were already set at these settings, or select ‘apply’ if the settings needed to be switched to the correct settings.

Recording

1. Line up the well with the frame that can be seen on the screen and ensure that the well is fully focused.
2. Check that the Filename ends with the index/number of the plate and well that is being recored.
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When switching between timepoints or plates, click on the film roll icon under ‘Filename’. Change the Filename prefix to the correct timepoint or plate and change the Index back to 1.
3. Click the red dot icon to begin recording the well, which will record for 15 sec.
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When one well is filmed the filename should change by one number for recording the next well
4. Move the 96-well plate to the next well and repeat steps 1 - 3

Cleaning up

1. Plug in the portable external hard drive into the open USB port.
2. Click on the icon for the hard drive found on the desktop to open up the files in the hard drive.
3. Drag the folder from the desktop into the hard drive files which should make a copy of the folder into the hard drive.
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Double check that both the desktop and the hard drive contain the videos that were filmed that day, and all videos have completely copyied over.
4. At the bottom right of the desktop, click on the USB icon in the toolbar, select the hard drive, and click ‘Eject’.
5. A message will appear when it is safe to remove the hard drive from the USB port.
6. Exit out of all the programs and folders open on the desktop.
7. Sign-out of the computer.
8. Push in the bottom knob on the right side of the stand, to turn off the transmitted light.
9. Flip the switch on the backside of the camera from left to right.
10. Unplug the USB cord for the microscope camera from the computer.
11. Remove anything from the base of the microscope.
12. Replace the blue cover over the microscope.

Analysis

All analytical ImageJ macros can be found in the associated Wheeler Lab GitHub repository:

ImageJ-macros

wheelerlab-uwec ⋅ a year ago