BZ-X800 Image Acquisition Settings
CODEX®-specific settings for the Keyence BZ-X800.
Overview
CODEX® Multicycle experiments require specific Keyence BZ-Viewer software settings to be enabled. Specifically, the Channel and Camera settings, Z-stack, Regions, Auto-Focus, and Auto-save settings have to be enabled in the Keyence BZ-Viewer for integration. This chapter describes what needs to be enabled for each setting. For details on the specific operation of each of these settings within the Keyence BZ-Viewer, refer to instructions provided by Keyence.
The figure below outlines all the settings that needs to be configured in the Keyence software, and the following subsections provide the necessary details for each setting.
Channels
CODEX® Instrument Manager software (CIM) expects four enabled channels and in a specific order as shown below. If the four designated channels are not enabled or turned on, the software will not detect the channels.
The user sets the filter cube names on the Keyence software. See the Keyence manual for instructions on how to name or change the names of the filter cubes.
Ensure filter cubes have been loaded in the proper order depending on tissue type and/or the Reporters used in the experiment. We recommend that the following channels be used - DAPI, AF488, ATTO550, and Cy5 for Fresh Frozen tissue samples, and DAPI, ATTO550, Cy5, and AF750 for FFPE tissue samples.
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 all 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:
Auto-Focus
When selecting the central auto-focus location for any tissue, there are three factors to ensure the best automated focusing:
Tissue present
Dark areas around the tissue present
No debris in the Auto-Focus frame
CIM uses the first channel (DAPI channel) to Auto-Focus the sample. The Driver software uses the Move Down button to lower the objective, and then Auto-Focus button to 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 Keyence software to take images of the tissue across multiple Z-planes. To enable Z-stack ensure Z-stack is selected. Per tissue, you will set the number of Z slices being taken per tile and the distance (pitch) between each slice (each image).
The Z-stack limits are set by the CIM software during the beginning of each image acquisition cycle. The Pitch is set by the user, and is recommended to be 1.5 um.
Within CIM, the total Z range is set as steps of 1.2um. (Note: Keyence only allows setting the Z-range in 1.2 um increments). With a pitch of 1.5um, this does not always result in the exact expected number of Z-slices.
On the experiment screen, CIM will show the estimation of final number of Z-slices as shown the snapshot below
It is recommended to use an odd number of steps, this will ensure the focal plane is in the middle. This ensures an equal number of 1.2 um steps above and below the focal plane.
Increasing the number of slices results in increased captured images and increased run time. We recommend a pitch of 1.5 um with 9 slices for good quality data from 5-10 um thick tissues. This can be achieved by using 11 1.2um slices.
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 sets up sample regions using both the (A) Stitching and (B) 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. The Navigation window sets the center of each region. A new region is added by clicking Add. Within the drop down, every Created option will be imaged as a separate region.
For acquisition of more than 10 regions at a time, please refer to the Multipoint Acquisition settings
Data saving settings
Keyence software saves the data as it is being written. For every new region and cycle a new folder is automatically created by the Keyence BZ-Viewer software .
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®. This allows to specify the folder for acquired data. By default, 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 software 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.
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