Cresyl Violet LM Imaging

Owned by Everett I Owens
Last updated: Jan 30, 2024 by Adrienne Li
5 min read


  1. Turn on computer, monitor, and microscope. 
  2. Open the “ZEN” application with circular icon, select “ZEN Pro.”
  3. Wipe both sides of slide with Kimwipe and orient it on stage,
    1. Label down
    2. White strip on right side
    3. Section numbers will read from right to left, meaning when looking down at the slide the first section is in the top right while the fourth section is the one below it
    4. Ensure that first section is directly above the objective by using the stage controller (one knob controls left-right movement, the other controls forward-backward movement)
  4. Set up microscope,
    1. Physically turn the objective wheel to the brown 2.5x magnification objective 
    2. Be sure the light wheel above the stage is turned to “H”
    3. Set 20% light intensity:
      1. “Locate” tab in the top left on ZEN -> “Microscope Control” drop-down -> select the top light bulb and enter “20%"
    4. Toggle the wheel on the left side of the base of the microscope to show an arrow (meaning it’s directing output to computer rather than to eye piece)
  5. Set exposure,
    1. “Locate” tab on ZEN -> “Camera” drop-down
      1. Set “Exposure Time” to 0.420 ms
      2. Set “Exposure Time Intensity” to 75%
  6. Bring specimen into focus,
    1. Navigate to “Locate” tab -> select “LIVE” -> zoom in using mouse wheel -> use microscope knobs to maximize focus -> zoom back out
    2. End live feed by clicking “Stop” and clear the .czi file from the bin in the top right by clicking the “X” (it’s a photo from when you started the LIVE feed)
  7. Take panorama of the first section,
    1. “Locate” tab -> select “Live Panorama” drop-down -> click “Start Live Panorama”
    2. Use the stage controller slowly to take full panorama of the section
      1. May be helpful to zoom out using the mouse wheel, or by enabling “Auto Fit” from options menu below the microscope image
      2. Ensure the entirety of the section is included in the panorama
      3. Click “Stop” when done
    3. Select “Yes” when prompted about image generation after panorama is complete
    4. The .czi file of the panorama will be displayed on screen, as well as stored in the “Container” in the top right of the application.
    5. Use stage controller to move the next section over the objective.
  8. Repeat Step 9 until each section on the slide has been captured in a panorama *be sure you go in order according to section number, as mentioned in Step 4c*
  9. Save each of the panoramas,
    1. Double click on the first image in the container
    2. Shift+left click on each of the .czi files in the container
    3. Right click on the selected items and choose “Save Selected As…”
    4. Pictures -> Synpo -> that specific Specimen’s name folder -> CZI Raw
    5. You will be prompted to save each of the panoramas from the “Container”
    6. Format of the file names should be “specimen name_slide#sec#_raw”
      1. eg. "SP6B3(4)_13_slide14_sec1_raw”, with the section number increasing by one each time you are prompted to save
  10. Stitch the panoramas
    1. Navigate to the “Processing” tab
    2. Select “Batch”, instead of “Single”
    3. Reselect each of the files from the container and drag them into the “Batch Processing” area, where it says “Select a function first….”
    4. Highlight the files under “Batch Processing”-> click the “Batch Method” drop-down -> select “Stitching” (each of the files should now read “Stitching” under the “Method” column)
    5. Click the “Parameters” drop-down
      1. Edge Detector - no
      2. Minimal Overlaps - 5%
      3. Maximal Shift - 10%
      4. Comparer - Optimized
      5. Global Optimizer - Best
    6. Unselect “Use Input Folder as Output folder” in the “Batch Processing” area, click the adjacent three dots, and select “CZI Stitched” folder (Pictures -> Synpo -> Specimen name -> CZI Stitched) as the images’ destinations
    7. Ensure that each file in the “Batch Processing” area have identical “Methods” (Stitching) and “Output Storage Path” columns, and then click “Apply” on the upper left
    8. Wait until all files have check marks on the left side
  11. Export images as .tif files
    1. While still in the “Processing” tab, click “Remove all"
    2. Open File Explorer, and locate the stitched .czi’s you just saved (Pictures -> Synpo -> specimen name -> CZI Stitched)
    3. Select all of the stitched .czi images and drag them into the “Batch Processing” area
    4. Highlight them and choose “Image Export” from the “Batch Method” drop-down
    5. Under the “Parameters” drop-down, click the bar next to “Settings” select “TIFF Processing”
    6. Unselect “Use Input Folder as Output Folder” in the “Batch Processing” area, click the adjacent three dots, and select “TIF Stitched” folder as the images’ destinations
    7. Ensure that each file in the “Batch Processing” area have identical “Methods” (Image Export) and “Output Storage Path” columns, and then click “Apply”
  12. To continue,
    1. Clear the “Container” by selecting all items and clicking the “X”
    2. Click “Remove All” from the processing area
    3. Replace the slide, and repeat from Step 7
  13. When done, power off computer, monitor, and microscope, and cover the microscope

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