TITLE: Commercial MIBI Walkthrough: Protocol &amp; Instructions
AUTHORS: Bryan Cannon, Christine Camacho

[DESCRIPTION]
This is the summary protocol for operating a commercial MIBI in the Bendall & Angelo Labs.
Will be updated as new internal and external features are adopted.

[STEPS]
SECTION: Pre-check
4. Unless actively navigating your tissue or slide, make sure to keep the MIBI Control in STANDBY mode (not SED or SPOT)

   

SECTION: Pre-check
1. Ensure the machine you’re working on (P9, M13, M16) is currently UP on the machine status sheet and slack channel #mibi_instrument_status.

 
SECTION: Pre-check
1.1. NOTE: If you encounter an issue with your run and need to send a ticket to Ionpath (support@ionpath.com), make sure to update the sheet with the machine as DOWN and cc Mako (makogold@stanford.edu) & Christine (christinecamacho@stanford.edu) so they can sign the machine back to UP status after resetting the appropriate imaging settings once Ionpath has finished. 
SECTION: Pre-check
3. Log into MIBI Control with your account (upper right hand corner of UI)

  
 

SECTION: Pre-check
2. Check available space on machine’s D drive, named MIBIData (ensure there is at least 50GB on drive free for non-cohort runs)

  

SECTION: Quality Control: PMMA Counts
10. If running a PMMA, you can use the instructions below with the PMMA slide assigned to each machine.

Load PMMA slide and move stage to a position close to previously imaged PMMA FoV.
Switch Mode to SED.
Alter Detector Gain to see image.
MAKE SURE NO FOVS ARE LOADED AT THIS POINT OR CHECK WILL OVERWRITE.
Set up FOV - 512 x 512, 200um, 0.5 ms, 1 scan, Imaging mode: Coarse, Target: PMMA slide.

 
SECTION: Sample Exchange
6. One slide at a time - press Exchange Sample in MIBI Control, and confirm. Take the current slide out of the slot further from you and put your slide into the slot closest to you.

  

SECTION: Choosing Points
12. Tiling / TMA Tool: Highly recommended for longer runs
Tsai Lab tool for tiling on Optical or SED images
  
Further notes of use are here in the extended protocol.

SECTION: Focusing
14. You need to alter the focus and stigmation of your image before starting your run.
Go to area of tissue not used for acquiring.
Switch Mode to SED, Imaging Mode to whatever setting you set your points to.
Starting with a large FOV, alter focus and stigmation until center is clear, top/bottom are slightly blurry, and repeat for smaller and smaller FOV windows (e.g. 400 and 100 um) at your acquisition resolution.
Each time you finalize an FOV’s focus settings, make sure you hit SAVE.

  
 

 
SECTION: Acquiring Data &amp; Monitoring
15. Load back FOVs from exported json file.

 
SECTION: Post-Run
16. Set the machine to STANDBY mode in MIBI Control.
SECTION: Quality Control: Molybdenum Foil Counts
11. Run a single moly FoV to check general counts, should do this before every run.

Change ‘Target Selection’ to select Molybdenum Foil.
Switch Mode to SED.
Alter Detector Gain to see image.
MAKE SURE NO FOVS ARE LOADED AT THIS POINT OR CHECK WILL OVERWRITE.
Set up FOV - 128 x 128, 200um, 1 ms, 3 scans, Imaging mode: QC 100um, Target: Molybdenum Foil.

  
SECTION: Choosing Points
13. Individual Selection
Switch Mode to SED, Imaging Mode to QC 300.
Click on area of slide and push ‘Move Stage to Target Position’ button for large movements.
Use ‘Jog Stage’ popout for larger movements.
Single arrow = single increment denoted by number in window.
Double arrow = 10x movement of number in window.
Triple arrow = 100x movement of number in window.
After finding a spot, click on ‘Add FOV’.
Enter parameters: resolution, FOV size, dwell time, imaging mode, etc.
Give the point a name and assign it to a slide / section.
Save.
Move to next point and repeat until all points a re selected.
Export FOVs as a json file (will need to import later).

  

SECTION: Pre-check
5. Make sure Lens 1 voltage where it should be in HV Control Chrome tab: ~505V.  
  

SECTION: Pre-check
5.1. DO NOT Change - talk to Mike, Marc, Christine, or Mako if it shows a different value.
SECTION: Sample Exchange
6.1. Make sure the slide loads properly; the image should load in the left window. Assign slide ID as created in Ionpath MIBItracker under your account.
 

SECTION: Quality Control: PMMA Counts
10.4. NOTE: PMMA slide is used for checking counts distribution from channels we will actually extract from. 

Ideal to run before loading a slide during cohort runs, ensuring signal distribution is normal. 
Do NOT do this between separate runs on the same slide, e.g. if setting up multiple runs on a single slide with a large TMA.
SECTION: Quality Control: General
7. Current check: confirm imaging mode current values match expected output.
Detector Calibration: To maintain a consistent ion response, or image brightness, the detector gain is calibrated by measuring Molybdenum Foil counts over a range of gain settings. 
Molybdenum Foil check: confirm built-in, general counts acquired at a specific current.
PMMA check: confirm specific counts at mass ranges of interest at a specific current.
Ionpath Quick start guide outlines more specific details as needed.
SECTION: Quality Control: Molybdenum Foil Counts
11.2. Record values in log sheet.

 
SECTION: Quality Control: Detector
9. Detector Calibration
Press button, and MIBI Control will begin a detector sweep.
MAKE SURE NO FOVS ARE LOADED AT THIS POINT OR CHECK WILL OVERWRITE.

 

SECTION: Quality Control: Current
8. Check current values for consistency and accuracy.

Change ‘Target Selection’ to select Faraday cup.
Switch Mode to SED.
Alter Detector Gain to see image.
Turn on image crosshairs and use jog to center cross to the middle of the cup.

  

SECTION: Quality Control: PMMA Counts
10.3. If values are NOT massively different from previously recorded values (i.e. 20% difference okay, ~log difference not okay) then pass.
If values are problematic, ask other lab members and/or Mike.
SECTION: Quality Control: Current
8.1. If acquiring in Coarse mode: Change Mode to SPOT, Imaging mode to Coarse.
Sample current - want around 9.5.
Change Lens 1(V).
Record values in log sheet.
SAVE to Imaging Mode.

   
 


SECTION: Quality Control: Current
8.2. If acquiring in Super Fine mode: Change Mode to SPOT, Imaging mode to Super Fine Imaging Mode.
Sample current - want around 2.5
Change Lens 1(V).
Record value in log sheet.
SAVE to Imaging Mode

   

SECTION: Quality Control: Molybdenum Foil Counts
11.3. If values are NOT massively different from previously recorded values (i.e. 20% difference okay, ~log difference not okay) then pass.
If values are problematic, ask other lab members and/or Mike.
If running for longer than 24 hours:
- Monitor MPH, if below a certain value, detector gain will need to be boosted.
- AutoGain will eventually handle this. TBD
SECTION: Quality Control: Molybdenum Foil Counts
11.1. Start Run.

Integrate Moly 98 and Moly 92 counts for the 3rd scan. 
Use the GUI to enter in the integration values (97.7-98.3, 91.7-92.3, respectively)

 
SECTION: Sample Exchange
6.2. Enter your run, date, and slide info into the log sheet

  

SECTION: Quality Control: PMMA Counts
10.2. Record values in log sheet under the "PMMA" tab.     

SECTION: Acquiring Data &amp; Monitoring
15.2. Look at your images in the Ionpath MIBIViewer.
 
    

SECTION: Quality Control: PMMA Counts
10.1.  Start Run. 

Integrate values for each PMMA peak (89, 113, 142, 145, 150, 159, 176).
Use the GUI to enter in the integration values from -0.3 to 0 (e.g. 88.7-89).

 
SECTION: Focusing
14.1. Write down all numbers in log sheet.

 
SECTION: Focusing
14.2. When done, put SED back into standby mode so you don’t ablate your tissue sample.
SECTION: Acquiring Data &amp; Monitoring
15.1. Start Run. 
Observe first few points in GUI for channel dropouts, low counts, any focus issues.
SECTION: Quality Control: Detector
9.2. If the calibration fails, up Detector voltage in HV control (bottom of page) and run again. If subsequent calibrations fail, then talk to Mike, Marc, Christine, or Mako.
SECTION: Quality Control: Detector
9.1. If the calibration passes, record detector value from HV Control (top image) in log sheet (bottom image)