TITLE: CODEX® Multiplexed Imaging | Microscope Setup and Tissue Imaging
AUTHORS: Diane  Saunders, Conrad Reihsmann, Marcela Brissova, Alvin C. Powers

[DESCRIPTION]
This protocol describes the imaging workflow currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group for CO-Detection by indEXing (CODEX®, now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.

[STEPS]
SECTION: Experiment Preparation
1. Check and power up equipment: CODEX® Instrument and Keyence BZ-X810 microscope.


  
SECTION: Experiment Preparation
4. Prepare 1 L of 1X CODEX Buffer (100 mL 10X CODEX Buffer in 900 mL MilliQ/ddH2O).
SECTION: Experiment Preparation
5. Fill bottles for the CODEX Instrument accordingly:

Bottle 1 - 1X CODEX Buffer
Bottle 2 - DMSO

⚠ Make sure the Waste Bottle is empty.
SECTION: Experiment Preparation
7. Launch the CODEX Instrument Manger (CIM) software. Load or create an experiment template and input appropriate labels under Project, Experiment, and Operator fields.
 

SECTION: Experiment Preparation
8. Specify the number of cycles in Total #Cycles and enter the Start Cycle Well (the first well in the reporter plate for the experiment).
SECTION: Experiment Preparation
9. Double check template contents, including marker names, classes, and exposure times. Set Z-Stack Planes to 5.
SECTION: Experiment Start Wizard
11. Prime the instrument: 

Briefly soak a gasket in 1X CODEX Buffer. Load a blank coverslip onto the stage plate and place gasket on top. Fit the stage insert, tighten, and place the assembly on a horizontal surface (in a plastic collection container or on the benchtop). Click Prime on the Experiment Start Wizard.
SECTION: Experiment Start Wizard
12. Load sample: 

Once the machine is finished priming, remove the stage insert and gasket and discard the blank coverslip. Carefully load the sample coverslip into the stage plate, place gasket on top, and tighten the stage insert. Remove any buffers or salts from bottom of the coverslip using a Kimwipe dipped in water. Next, situate the stage plate/stage insert into the holder of the Keyence BZ-X810 microscope, making sure fluidics lines can exit through the front of the microscope.



    
SECTION: Experiment Start Wizard
13. Add DAPI:

Pipette 700 µL of Nuclear Stain Solution (0.067 % (v/v) Nuclear Stain in 1X CODEX Buffer) onto the sample coverslip. Click Immediately Wash Tissue on the Experiment Start Wizard.
SECTION: Post-Run
17. Once run is complete, carefully remove coverslip and place back into Storage Buffer for preservation at 4 °C if future imaging or staining is to be performed. Otherwise, coverslip can be discarded.
SECTION: Post-Run
18. Load a blank coverslip and perform a Clean Instrument Wash by navigating to the Maintenance tab. If another run will not be performed immediately, place Bottle 1 and Bottle 2 lines in a beaker of MilliQ water and perform a Maintenance Wash.
SECTION: Experiment Preparation
10. Press Start Experiment button and follow the guided Experiment Start Wizard prompts as outlined below.
SECTION: Experiment Start Wizard
15. Microscope Pre-Check:

Back in CIM Experiment Start Wizard, select Microscope Pre-Check. If pre-check fails, double-check microscope parameters outlined above and in the Quick Reference Guide.
SECTION: Experiment Start Wizard
16. Press Start Run. Stay several minutes to ensure sample well is not leaking before walking away.
SECTION: Experiment Start Wizard
14. Configure microscope settings:

Minimize the CIM window and Experiment Start Wizard and launch the BZ-X Viewer software. Choose Capture Still Images, and select Versatile as the Sample Holder.
SECTION: Experiment Start Wizard
14.1. Verify the following settings:

Set to Multi-Color
Enable all four fluorescent channels (DAPI, GFP, TRITC, Cy5)
Set all channel pseudocolors to white
Set Excitation Light to 100% and enable Low Photobleach
Set camera to Mono
Set Resolution Sensitivity to High Resolution

In Options tab:

Select Z-Stack  -> Multi-Color
Enable Gain and Transmitted Light Power
Enable Excitation Light Filter
Choose 14bit under TIFF Image Setting
Enable Automatically Save Image and set Format to TIFF

Under Capture Area Settings:

Enable Z-Stack
Enter 1.5 μm for Pitch
Enable Stitching
Choose Set Center and Number of Images
SECTION: Experiment Start Wizard
14.2. Set Z-stack parameters:

Ensure that the tissue is situated over the objective, moving the stage using the BZ-X Viewer as needed. Switch to the DAPI channel and perform autofocus to locate the tissue. At multiple points across the tissue, find and note the optimal focus (Z position). If values are within ~5 μm of each other, scroll to the center Z position and under Capture Area Settings, select Fix Range and click Set. Range should correspond to 5 images (Z-stacks), approximately ~6 μm.


  
 

SECTION: Experiment Start Wizard
14.3. Set imaging region:

Bring up the Navigation window, adding regions as needed to locate the edges of the tissue. Use these macro stitched images to approximate the center of the tissue and click Add.  Define region size by setting the number of X and Y images (tiles) under Capture Area Settings. Without changing positions, scroll back through stored regions and ensure that the green box (denoting the captured area) includes the tissue edges. Adjust center and/or X and Y numbers as needed.


 
 
Once you are satisfied with the imaging region, delete all other regions stored in the Navigation window so that only the central region remains. Select Start Capture and specify the file path for images to be saved (folder should match Experiment name). Press Stop after a few images.
SECTION: Post-Run
19. Empty the vacuum waste container and small solution containers under the hood. Rinse, and leave to dry on paper towels. Remove reporter plate.
SECTION: Experiment Preparation
6. Load the prepared reporter plate, noting the coordinates of the first well containing solution.
SECTION: Post-Run
20. Shut down BZ-X Viewer and CIM software.
SECTION: Experiment Preparation
2. Equilibrate the following to Room temperature :
Coverslip in storage buffer
Prepared reporter plate
SECTION: Experiment Preparation
3. Gather additional supplies: