Experiment

Experiment set up and run CODEX® experiments with the CODEX® Instrument Manager (CIM)

Overview of Experiment Tab

The CODEX® Instrument Manager "Experiment" Tab is used to set up and start a CODEX® run.

Database and Templates

The CODEX® Instrument Manager (CIM) uses the concept of ‘templates’ to create an experiment. The project’s experiment conditions can be created once to be used over and over again over the course of the entire project.

• Upon installation, an editable sample template is available. The template files are XML files.

• The first step of the creation of an experiment is to load a template.

• Experiment changes can be saved to the same, or a new template

• CIM will make sure to ask for saving the template on any changes.

• The XML files can be easily shared between CODEX® instruments for sharing parameters.

• Note that the information collected and stored during CODEX® experiments is stored elsewhere. Templates are only used for creating an experiment.

Opening a Template

Click on Open or New Template to create an experiment design based on that template

• Open Template -This button allows users to open a saved Template

• New Template - This will open a default experiment. All Marker names will be labeled by both channel and cycle. This new experiment can be saved to a new Template file.

Saving the Template

CIM allows users to Save the template as is or Save As for renaming of a prior different experimental setup.

Experiment Screen

The table, within the Experiment Management tab, enables full control and visibility of experiment parameters during the run. Read this section to understand each of the functions within the design table.

Experiment design

The grid within the experiment design screen represents the parameters of the study.

Each row represents one well on the 96-well Reporter plate. Every well contains the Nuclear Stain, which will be captured in the DAPI channel. One Reporter per channel can be revealed in a given cycle. This is determined in the Reporter plate setup in Chapter 7 in the user manual.

Naming

CIM allows for both Project and Experiment naming

This feature allows for tracking of both the individual runs and the projects.

Note: Experiment and Project Name cannot include the character "#".

Table Options

Example of 4 cycles starting from well A1

• Include - When not using Skip Wells this column is empty. When using Skip Wells the check mark determines if the well is used for the experiment.

• Cycle - The cycle number. Depending on the Microscope used, the image data files will be saved in folders (Keyence, Leica), or CZI files (Zeiss) per cycle. This number will correspond to the Reporters/Markers present in a given cycle.

• Well - The physical location on the plate. This cannot be edited within the table. To change the starting well, select the Start cycle Well. Individual wells can be skipped.

• Channel Options - These are options set for each Channel.

  • Marker - This editable field contains the antibody name. This name is used within the Analysis software.

  • Class - This attribute defines the well class (Nuclear Stain. Marker, or Blank) The class affects how wells will be analyzed.

    • Nuclear Stain

    • Marker

    • Blank

    • Empty

  • Exposure - This is the exposure time set for the given marker, in milliseconds. This will be microscope and biomarker-specific. See the Microscope Manual(s) for more information.

Start Cycle Well

Example of start cycle well dropdown

The experiment run selects the dyes from the plate in order by row. Select the first well in which the Reporters have been physically placed within the table.

The Well name will change in the table automatically, in order by row. This enables the same experiment parameters to be used over and over again regardless of the starting location of the Reporters within the plate. The Well name cannot be edited within the table.

Channel Assignment

Channel Assignment selection button

Users can set the order of the four channels. There are five channel options: DAPI, AF488, ATTO550, Cy5, or AF750.

DAPI must always be one of the options. For most microscopes, the first channel is the recommended DAPI channel.

Select OK for the changes to take effect.

The order of the channels in CIM should correspond to the order of the channels within the microscope software!

The Channel designation pop-up.

Cycle order and skipping wells

Cycles run contiguously by row. If there ever is a well that needs to be skipped, select the Skip Well option.

Skip Wells button

This selection will give you the ability to deselect wells from the run. When a well is skipped for a run, the row will turn grey.

A full table view when a well is skipped.

Validate Experiment

The Validate Experiment button will check the table for any user inputs that may be incongruous with the experiment. For example, if a letter was typed into the exposure time option.

Opt to receive an email notification at the end of each cycle

With CIM 1.29, users can now choose to receive an email notification at the end of each cycle.

Before starting a CODEX run, select the following option available on the experiment setup tab

Enable Sample hydration at the end of an experiment

CIM 1.29 allows users to enable sample hydration cycles at the end of an experiment, in case the sample needs to be preserved for downstream experiments or a second CODEX run

Selecting the following option on the experiment setup tab before initiating a CODEX run will enable hydration cycles every 90 mins for up to 48 hours at the end of an experiment

Option to automate Keyence-software-enabled stitching of raw data

CIM 1.29 allows users with Keyence integration to automate the stitching of raw image data at the end of an experiment. The stitching is enabled by the Keyence software and the raw stitched data can be used to review and analyze the results prior to data transfer and processing.

End of Experiment options

At the end of a CODEX run, the users can select one of the three options:

1. Clean the instrument: It is highly recommended that at the end of each experiment, the instrument fluidic lines are cleaned. Upon selecting the "Clean the instrument" option at the end of an experiment, "Clean instrument" protocol is initiated.

If the user would like to save the sample, prior to choosing the "Clean Instrument" option, please make sure to replace the Sample coverslip with an Empty coverslip

2. Add one or more cycles: The user can choose to add one or more cycles to the experiment and continue with the experiment.

It is critical to make sure that the experiment cycle setup aligns with the 96-well well reporter plate and that the appropriate wells are selected.

3. Finish experiment:

If the options 1 or 2 are not applicable, the user can select "Finish experiment" to exit out of the experiment,

It is highly recommended that the instrument is cleaned by selecting Option #1 "Clean the instrument" at the end of an experiment.