Image Acquisition Settings
Setting up Zeiss for CODEX runs.
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.
Zen Blue 2.3, 2.5 or 2.6 is needed to run CODEX.
The figure below outlines all the settings to be enabled in the Zeiss software. The following subsections provide detail for each setting.
Options
To see the options you need for Zeiss, select the below.
Z-Stack
Tiles
Full Z-Stack per Channel. Do not set All channels per slice.
Auto Save
Channels
CIM is looking for four channels. If there are not the designated number of channels, the software will not work. The channels order with Zen Blue should match the CIM Experiment table order.
Channels can be selected and added from a list with the Zeiss software by clicking the ‘+’ sign. It is important the channels are in the proper order and that a checkmark is selected only next to the channels being used. The excitation percentages are set for each channel by selecting the channel.
Please determine the optimal excitation percentage before setting up for a CODEX experiment.
We recommend excitation percentages of:
DAPI: 20 - 35%
FITC, TRITC, Cy5: 50-100%
Regions
Akoya refers to the section of tissue that is imaged as a region. Single or multiple regions can be collected on a coverslip. CODEX analysis software only supports imaging of rectangular regions. Polygonal regions will be enabled in a future release.
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
Currently, only multiple RECTANGULAR regions of the same size are supported.
Zeiss refers to the imageable regions as Tilescans. The Tiles tab enables both setting of a region and a list of regions (A). First, find your sample, focus it, and do a general preview scan. To draw over specific regions, go to the Advanced Setup (B). Within the bottom pane, use the square control tool to draw regions over the tissue. Each region will show up in the main tab. Here is an example of two regions selected (C).
Autofocus
General
When setting the central autofocus location of any tissue there are three factors to ensure the best automated focusing.
Tissue present
Dark areas around the tissue present
No debris or bright spots in the autofocus frame
Autofocus Steps
Zeiss offers several options for autofocusing the sample. Akoya recommends the Focus per Tilescan option when using Zeiss. A majority of the options are set within the Focus Strategy tab. When setting up an experiment, it is a combination of 3 or 4 settings that need to be in place to allow for optimal autofocusing. It is recommended to find the optimal autofocus settings before a CODEX run.
The autofocus steps are:
Set the Reference Channel in the Channel tab
Set the tile focusing options in the Focus Strategy tab
Set the Z-map from within the Tiles tab
(Optional) Definite Focus if hardware is available and wish to use it
1. Setting autofocusing channel in Zen Blue
The Zeiss refers to the autofocus channel as the Reference channel. To set DAPI as the reference channel, right click on DAPI to select the appropriate option. DAPI will have the term ‘Ref.’ listed next to the display color option if set correctly.
2. Set the Focus Strategy
Akoya recommends to use:
Use Focus Surface/Z Values defined by Tile Setup
Local (per Region/Position)
Adapt Focus Surface/Z Values
By Tile Setup
For the Stabilization Event Repetitions and Frequency, select Expert to see all the options. There are four general options that can be used. These options are sample dependent.
Tile Regions: Can set this to repeat for a certain number of tiles over the entire region Center of Region First tile of Region
Every other tile
Center of Regions
First Tile of Regions
3. Set the TileScan Focus
Within Tile setup (A). Select ‘Verify Tile Regions…’ After you click verify tile regions, a new pop up appears (B).
Set the dropdown to AutoFocus next to select Verification Helper Method.
Click Move to Current Point. Confirm and set Z.
Click Run AF and Set Z Click
Use AF to Verify the Remaining
It is important that ‘Include Z when Moving to Points’ is selected.
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.
To enable Z-stack, check the Z-stack box at the top of the screen (A).
The Z-stack settings are set within the microscope software not within CIM (B). Set the total tissue Range (depth) and the Interval (pitch) in the Zen software. Once the range and interval are set and the current z slice is the best focus, select ‘Center’. The number of steps/slices is set in the CODEX Driver.
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.
Data saving settings
CODEX® requires autosaving your data so nothing will be lost within the experiment. Zeiss saves each cycle as a CZI file. Every region is saved together per cycle.
Under the Auto Save menu, make sure Automatic Sub-Folder is selected and choose a name.
CIM does NOT support the use of comma (,) in the experiment path. Please make sure there are NO commas in the "Folder" tab.
Camera settings
The camera settings must be set correctly to capture the full dynamic range and allow comparison between images.
Ensure that the following settings are selected:
Image Quality | Shadow | Highlight | Gamma |
16-Bit | 0 | 255 | 1.0 |
CODEX specific settings
ZEISS enables users to save and load previous settings. After selecting all the settings in this chapter, save your settings. You can also clone your settings.
Note that if there is an asterisk next to the name of the file, settings have been changed. Zeiss will save the Tile scans within these settings. After every experiment setup, there still therefore be an asterisk next to the name of the program file.
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