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Image Acquisition Settings

Overview

The CODEX® multiplexing requires specific LASX settings to be enabled. Specifically, it needs the channels, camera settings, Z-stack, Regions, autofocus, and autosave settings enabled in LASX in order to function. This chapter will describe what to enable for each setting. For specific operation of each of these settings within LASX, refer to Leica instructions.
LASX 3.6 or LASX 3.7.1 is required to run CODEX. Lower versions will result in focusing issues during acquisition.
The figure below outlines all the settings to be enabled in the Leica software. The following subsections provide detail for each setting.
LASX3.6

Data saving settings

CODEX® requires autosaving your data so nothing will be lost within the experiment. Each CODEX Cycle is saved as a Project. In order to save all CODEX data correctly, the Project must be setup prior to any other microscope configuration setup.
To start a new experiment save location, go to the Open Projects tab.
Remove any existing projects before creating any new projects.
Set the Save settings by clicking on the + icon.
To set the file save settings,click on the ‘+’ sign next to Auto-Save, a new prompt box will appear.
  • Autosave: On
  • Data Type: TIF
  • Compression: Uncompressed
  • Shift to 16 bit: Unprocessed
CIM does NOT support the use of comma (,) in the experiment path. Please make sure there are NO commas in the "Project Location" tab.

Channels

CIM is looking for four channels. If these are not the designated number of channels, the software will not work. The channels order with LASX should match the CIM Experiment table order.
In the Image settings, set the FIM and IL-Fld. to full. Make sure the Light mode is FIM and the Camera % is 100%
Do not use QUAD LED excitation.

Autofocus

When setting the central autofocus 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 or bright spots in the autofocus frame
First, use the coarse focus to approximately bring the tissue in focus. Next, click the Autofocus button. The Leica will iterate through Z slices above and below the current Z plane to find the best slice. CIM uses the Autofocus button to autofocus the sample.

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 or z-step size (pitch) between each image.
Select Z to enable the Z-stack feature
Setting up Z-Stacks for 3 Z-Steps
Setting up Z-Stacks for 5 Z-Steps
Set the total tissue size in Z-size and the pitch, in Z-step size. The number of steps is indirectly selected after entering these two numbers.
Z-step size must be selected for proper CIM function.
To enable Z-Stack ensure Acquisition Mode is set to x-y-z . Make sure the Start and End buttons are clicked and red under the Z-Stack menu. Autofocus and then set the Z-step size. A step size of 1.5 um is recommended. We currently recommend that 3-5 z-steps be used (in conjunction with Focal Map settings) to minimize the effects of exposure of the sample to out of focus light. Increasing the number of slices results in increased captured images and increased run time.
Out of focus light, above and below the focal plane, can cause photobleaching of certain fluorophores. To minimize the exposure of the sample to light, we recommend using between 3-5 Z-stacks

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.
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 tissue microarray (TMA) – different regions each covering a single core sample
Leica DMi8 Multi-region datasets can be acquired by defining the region sizes within the LASX software. All regions must be rectangular and have the same z-slice settings.
Total region should not exceed 20x20 tiles.
A total of 25 rectangular regions can be acquired with this microscope configuration. When acquiring multiple regions, all regions must be the same size (e.g. all 3x3 regions or all 4x7 regions.... etc).
Leica uses the Navigation screen to set regions. The Navigation screen can be accessed under the Acquisition Mode menu (B). In the Navigation screen, a preview of the sample can be acquired with the spiral icon located at the bottom right. The preview will be acquired in the last channel that was enabled. To define a region of interest, click the rectangular selection tool (C). Imaging multiple regions using Leica is supported. Each region to be imaged will show up under the task list (D). All checked items in the task list will be imaged. Ensure that the autofocus box is inside the region (E). The autofocus box is the box representing the current field of view. It is where autofocusing will occur. A more detailed description of using the Focal Map feature is described below.
During microscope precheck and other CIM controlled microscope steps, make sure the Navigation Screen is closed.

Linked Shading

Leica offers a feature called 'Linked Shading' to correct for aberrations caused by the microscope's optics. Akoya strongly recommends using this feature before every experiment.
Move stage to Field of View without any tissue, artifacts or fluorescent signal. Confirm that region is blank using the DAPI channel. Open the Linked Shading menu.
On the Wizard tab, select 'Acquire'. The contrasting method should be FLUO. All settings, including the camera type, objective, exposure and z-plane should mimic, as close as possible, any settings that will be used during the experiment. At this point click Single Reference.
Once finished correcting for any shading, open the settings tab in the Linked Shading menu. To enable the shading correction that was just set up, check the box and confirm that the Linked Shading icon is now green.

Focal Point Mapping

Photo-bleaching artifacts are experienced in overlap regions, when certain dyes are used. We require that our users employ the use of Focal mapping to minimize the exposure of samples to light.
Focal Point Mapping allows for a large non-uniform area to be imaged with a limited amount of Z-planes. To use Focal Point Mapping for the CODEX experiment, enable the feature in the Experiment setup window of CIM. If it is not selected, CIM will not update the Z plane for each cycle. This will cause images to over time, come out of focus as the software will not compensate for the normal cycle to cycle drift compensation.
Highlighted in red is the 'Add Focus Point' button.
After creating your region(s) of interest, create focus points in region(s) by selecting the ‘add focus point’ option in the drawing toolbar in the Navigation window.
Example of a how Focal Points are plotted over a large tissue area (i.e. 11x11)
We recommend placing a focus point on every other tile of the region starting at the first tile imaged (top left corner). Areas without tissue do not need a focus point. Once all points have been placed, make sure the field of view is centered about the final tile with tissue (i.e. bottom rightmost tile for odd numbered and bottom leftmost for even numbered tiles).
Open up the focus map menu by clicking on the focus map icon on the toolbar. These focus points can be managed with the Focus Map option next to the drawing toolbar. All focus points made will appear in order of placement. By clicking the “...” icon next to Find all by Autofocus, users can increase the autofocus range that points will be found with.
Once the settings are at 40 microns and the most precise, users can close this menu out and continue to record z planes of each focus point by clicking “Find all by Autofocus.” After each point is found users can do a quick check to make sure all points are indeed in the best focus. If all points are in focus the focal map menu can be closed out and users can return to the main microscope settings page. The microscope check can be started at this point.

CODEX specific settings

Microscope software settings can be saved in the Leica software.
Under Load/Save single settings, choose save.

Additional Settings

These settings can be set once and forgotten.

16-bit

It is critical to set your Camera Preferences to 16-bit.

Tile Overlap

The percentage of tile overlap can also be set from the Navigation window of the Leica software. A 10% overlap is recommended for the best stitching results.