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BZ-X700 Image Acquisition Settings

CODEX specific settings for the Keyence BZ-X700.

Overview

The CODEX® multiplexing requires specific microscope software settings to be enabled. Specifically, it needs the channels, camera settings, Z-stack, Regions, autofocus, and autosave settings enabled in the microscope software in order to function. This chapter will describe what to enable for each setting. For specific operation of each of these settings within your microscope software, see instructions provided by the microscope company.
The figure below outlines all the settings to be enabled in the Keyence software. The following subsections provide detail for each setting.

Channels

CIM is looking for a set number of channels in a specific order. If there are not four designated number of channels, the software will not work.
The user sets the filter cube names on the Keyence. See the Keyence manual for changing and naming filter cubes.
Keyence can only have 4 filter cubes at a time. It is imperative to have the filter cubes be in the same order as specified in the CIM software.
Choose the Multi-Color setting. Under Channel Setting, make sure all channels are turned on. Keep the excitation light to 100% in illumination settings and select the low Photobleach option. Under camera settings, select monochrome.
It is important to set channels to Monochrome. The pseudocolor for all channels must be set to white for downstream processing.

Exposure times:

CIM will set the exposure time for each channel according to the experiment settings (in the Experiment designer).
Note that the experiment exposure times are set in milliseconds. CIM will calculate the closest fractional exposure time as used by the Keyence BZ-Viewer. This can result in rounding (above or below) the desired exposure time. Refer to the table here:
Fractions to milliseconds

Auto-focus

When setting the central auto-focus location of any tissue there are three factors to ensure the best automated focusing.
  1. 1.
    Tissue present
  2. 2.
    Dark areas around the tissue present
  3. 3.
    No debris in the auto-focus frame
Keyence uses the first channel to auto-focus the sample. The Driver software uses the Move Down button to lower the objective, and then Auto-Focus button to auto-focus the sample. Move the slider down with the ‘Move Down’ button and confirm the microscope can focus by clicking ‘Auto-Focus’.

Z-Stack

CODEX® utilizes the Z-stack functionality of the microscope software to take images of the tissue across multiple Z-planes. Per tissue, you will set the number of Z slices being taken per tile and the distance (pitch) between each image.
Keyence sets the Z-stack information through both the Driver software and the microscope software. The number of slices is set within CIM. The Pitch is set within the Microscope software.
To enable Z-stack ensure Z-stack is selected. Always set an odd number of Z-stacks in the CIM. Keyence works by setting the center number, then going a certain number above and below the center. If an even number is selected, the central slice will slightly be shifted but not imaged.
Increasing the number of slices results in increased captured images and increased runtime. We recommend a pitch of 1.5 um with 9 slices for good quality data from 5-10 um thick tissues.

Regions

Akoya refers to the section of tissue that is imaged as a region. Single or multiple regions can be collected on a coverslip. Keyence software only supports imaging of rectangular regions.
Examples of regions that can be taken:
  • Single region 1 tissue – a whole or partial tissue slice
  • Multiple region 1 tissue – different sections of the same tissue
  • Multiple region TMA – different regions each covering a single core sample
Keyence 700 Multi-region datasets can be acquired using the Navigation window dropdown window. Each region is defined by the dimensions outlined in the microscope settings tab (e.g. x=3 and y=3) and all regions must have the same dimensions and z-slice settings.
Each region will be autofocused and acquired in series to ensure proper image acquisition. Using this mechanism, there is a maximum of 10 regions that can be acquired and each region cannot exceed 25x25 tiles.
Keyence sets up sample regions using both the XY-Stitching settings and the Navigation window.
The Stitching window sets the X and Y tiles surrounding the center tile set in the Navigation pane by using the Set Center and Number of images X and Y. Each region will have the same tile size.
Example of a 5x5 tile
The Navigation window sets the center of each region. A new region is added by clicking Add. Within the dropdown, every Created option will be imaged as a separate region.

Data saving settings

Before starting a CODEX run, capture some images so the location of the data is set for the full CODEX experiment.
Keyence software saves the data as it is being written. For every new region and cycle a new folder is automatically created. When first setting up the data, it is critical to first save a couple of images in a specified path and folder and then stop before starting CODEX®. The reason for this is that the Keyence software will save in the last known location.
Please make sure during the experiment runs that each new experiment is placed in a separate main folder such that acquired images for a new experiment is saved within a new folder.
CIM does NOT support the use of comma (,) in the experiment root path. Please make sure there are NO commas in the "Root Path" tab.

Camera Settings

The camera settings must be set correctly to capture the full dynamic range and allow comparison between images.
Ensure image quality is set to 14 bit by selecting ‘Option’ (A and B). Ensure Shadow is set to 0, Highlight is set to 255, and Gamma is set to 1.0 (C).

Microscope settings - Pre-check:

Before starting an experiment, CIM will execute a ‘Precheck’. This will verify several key settings, and report the results of this ‘check list’. Although CIM scans several of the necessary settings, it does NOT scan all the required settings within the Keyence BZ-Viewer software. Hence, it is recommended that the user ensures that the settings are correct before starting an experiment run.