Multi-Point (BZ-X800)

For imaging more than 10 regions during a CODEX Run.

Multi-Point Acquisitions

CIM version 1.27 and above supports Keyence Multi-Point acquisition of up to 50 Points

Under Capture Area Settings, select all three options (Multi-Point, Z-Stack, and Stitching), as highlighted below:

Using the Navigation window, locate the first region of interest, and set the Upper and Lower Limits of the z-stack. Set the pitch (recommended 1.5μm).

Note: A Navigation region has to be added for pre-check to pass, but only registered points will be acquired.

Make sure to create the first region in the center of the sample, and this will be the point of auto-focus before image acquisition of each cycle. Hence, placing it in the center will give the best correction for drift. Additionally, it is crucial that this first point is a good quality region for proper auto-focusing.

Select “Set Center and Number of Images” and enter the X and Y dimensions of your ROI. To register the point settings, click “Set”. This registers the stage location for this ROI.

Navigate to the next region of interest, and select a new “stitched area” from the drop down menu:

Repeat the last two steps (i.e. Set region location & Region tile size) and click “Set” to register the Region. Regions can be of various sizes. Continue to register all ROIs of interest in this manner.

Considerations for Multi-Point over large regions

Keyence Multi-Point acquires data across all regions using the same z-slice settings. Thus, considerations need to be made when setting both the location of the first region as well as the number of z-slices.

With regards to the location of the first region, it is important to note that this region will define the auto-focus plane per cycle as well as the z-range that gets acquired. For this reason, it should be located towards the center of the total area defined by the group of Multi-Point regions.

With regards to the z-stack settings, it is recommended that the user selects more z-slices the larger the total area defined by the Multi-Point regions. When using Multi-Point, CIM software captures the same number of z-slices with the same pitch across all registered points. Since the CIM software will overwrite the Upper and Lower Limits set by the user in the Keyence software for Multi-Point, it is important to perform this calculation and then input the number of z-stack planes in CIM.

To empirically determine how many z-slices should be imaged, the user can find the highest and lowest points and calculate the number needed for a given sample using the following steps:

  1. After all regions are registered, identify the regions with the highest and lowest Z-positions.

  2. At the highest area, find the focal point, scroll up 2-5 Z-steps and record this Z-position (TOP).

  3. At the lowest area, find the focal point, scroll down 2-5 Z-steps and record this Z-position (BOTTOM).

  4. Go back to your first registered point and find the focal plane using Auto-focus and record this Z-position (FIRST).

  5. Subtract (FIRST) from (TOP) and divide by a pitch of 1.2 . This is how many z-steps above your autofocus plane that will be needed.

  6. Subtract (BOTTOM) from (FIRST) and divide by 1.2. This is how many z-steps below your autofocus plane that will be needed.

  7. Take the larger number of z-steps from Steps 6 and 7, multiply by 2, then add 1. This will the total number of z-steps (both above and below the first point's focal plane) that need to be imaged to ensure the highest and lowest portions of your tissue are in focus.

  8. Input this number of Z-stack planes into CIM.

Note: 1.2 is a fixed value for 20x objectives only. This is needed as the CIM will use the mouse actions to scroll up and down to set the Upper and Lower Limit with each acquisition cycle. The scrolling movements occur in 1.2μm steps for a 20x objective.

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