Keyence BZ-X810 All-in-One Fluorescence Microscope H&E Imaging Protocol

Setting up

1. Remove cover
2. Press power button on bottom right front of scope, light should turn blue
3. Check that the controller (box to the left of the scope) is on, it usually stays on
4. Open the BZ-X800 viewer program
5. Click “Capture Still Images”

Loading/changing slides

1. Click “Change sample”
2. Open the top panel of the scope and load in your slides, cover slip down. Be very careful, especially with unsealed slides, that there is no excess glycerol.
3. Take note of (or take picture on your phone) of where on the slide your samples are so you can navigate to them quicker
4. Close the panel

Locating sample

1. Click “live view” in the bottom right next to “Capture”. Instructions for each panel of the right sidebar of the program are as follows:
2. Lens
   • Select the 4X objective by clicking the drawing of the objective and selecting 4X, set zoom to 1X
3. CH Setting
   • Select CH4 BF by clicking on the channel name and turn it “ON” by clicking on the square to the right of the name.
     • This should be the only channel with “ON” selected
4. Illumination Setting
   • Select Transmitted Light and choose an illumination level. Usually between 50%-100% works well since we are not worried about photobleaching and allows for shorter exposure times.
5. Camera Setting
   1. Select Color
   2. Change Resolution Sensitivity to High Resolution
      • This is very important
   3. Change exposure using the slider or auto-exposure. You will likely need to redo this once your sample is in focus and regularly throughout imaging.
6. Microscope Tab
   1. Stage View (Right)
      1. Make sure the 3-slide sample holder is selected
      2. Scan for your sample based on your notes or picture where you think it should be approximately on the slide.
      3. It will be out of focus but you should be able to see some out of focus blobs of color if you reach the right area
   2. Z-position (Left)
      1. focus on your sample manually with the mouse wheel or with auto-focus
         1. fine adjustments: mouse scroll
         2. moderate adjustments: use ctrl + mouse wheel
         3. large adjustments: ctrl + shift + mouse wheel
7. Image Tab and Measure Tab
   1. All of these processing settings should be off for the real capture, but play around with them if helpful.
8. Navigation
   1. This can be really useful to save areas to come back to and to get quick overview scans of each section on your slide. Will add more detailed info.

Capturing tiled images

These settings are located in the bottom panel of the right sidebar.

1. Multi-Point should not be selected
2. Click the Stitching button
3. Select Set edge points
4. Move through all your edge points of interest and hit Set for all the corners of your capture area or any edges. It will automatically form a rectangle to include all your edge points
5. In the “Z-Stack” panel, check Auto-Focus Capture for CH4. Choose # of Z-slices based on how thick/complex your sample is
   • If all of the tiles will be around the same focus plane, you can use fewer slices.
   • NOTE: This will not capture a true Z-stack, it basically performs autofocus for each tile within a limited search range
6. Finally, go to a representative area of your capture area and focus the scope well. This will be the starting focal plane for the capture.
7. Start Capture
   • This will bring up a dialogue to save your files - it is limited to creating a new folder for each new capture, so keep this in mind

Saving tiled images + Scale metadata

This is super important but can be redone from the raw data saved in the folder on the scope computer if done incorrectly.

1. After the image is taken, click Analyze Images or double-click on the analyzer file in the folder where the images were saved
   • Only one tile will be shown in this first window, not the stiched image.
2. Click Load in the bottom right to open the Stitching Software
3. Image Stitch Window
   1. It may take a minute for this window to open
   2. Check “Correct Shading” with “Auto” selected for the Mode dropdown
   3. Select Uncompressed
   4. Check “Adjust Image Postions”
   5. Click Select Start Stitching
   6. When stitching is complete, the Wide Image Analyzer program will also open
4. Wide Image Viewer Window – where the image is acutally saved
   1. File > Save > Export in the Original Scale > TIFF
      • Can’t for the life of me figure out the Big TIFF thing.
5. Once the “Analyzer” window opens, hit Insert > Scale. In the window that opens, go to the bottom row labelled “Calibration” and click Setting...
   • WRITE DOWN the microns/pixel value for EACH tiled image.