Jun 19, 2023

Public workspaceCommercial MIBI Walkthrough: Protocol & Instructions

This protocol is a draft, published without a DOI.
  • 1Stanford University
Bryan Cannon
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Protocol Citation: Bryan Cannon, Christine Camacho 2023. Commercial MIBI Walkthrough: Protocol & Instructions. protocols.io https://protocols.io/view/commercial-mibi-walkthrough-protocol-amp-instructi-cttzwnp6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 05, 2023
Last Modified: January 31, 2024
Protocol Integer ID: 81497
Abstract
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.
Pre-check
Pre-check
Ensure the machine you’re working on (P9, M13, M16) is currently UP on the machine status sheet and slack channel #mibi_instrument_status.

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


Log into MIBI Control with your account (upper right hand corner of UI)



Unless actively navigating your tissue or slide, make sure to keep the MIBI Control in STANDBY mode (not SED or SPOT)



Make sure Lens 1 voltage where it should be in HV Control Chrome tab: ~505V.

DO NOT Change - talk to Mike, Marc, Christine, or Mako if it shows a different value.
Sample Exchange
Sample Exchange
  • 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.



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.
Note
The panel and slide ID must be entered and linked in the Ionpath MIBItracker prior to sample exchange.

Enter your run, date, and slide info into the log sheet



Quality Control: General
Quality Control: General
  • 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.
Quality Control: Current
Quality Control: Current
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.



  • 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.


Note
The sample current (nA) and lens 1 (V) have an inverse relationship. To decrease the sample current, increase the lens voltage incrementally.


  • 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



Quality Control: Detector
Quality Control: Detector
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.



If the calibration passes, record detector value from HV Control (top image) in log sheet (bottom image)



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.
Quality Control: PMMA Counts
Quality Control: PMMA Counts
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.


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).


Record values in log sheet under the "PMMA" tab.


  • 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.
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.
Quality Control: Molybdenum Foil Counts
Quality Control: Molybdenum Foil Counts
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.


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)


Record values in log sheet.


  • 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
Choosing Points
Choosing Points
Tiling / TMA Tool: Highly recommended for longer runs

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).



Focusing
Focusing
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.


Note
The stigmation (angle, magnitude) is uniform throughout the slide. The focus varies slightly along the y axis of the slide, i.e. the focus for a FOV towards the top of the slide will be slightly different than the focus for a FOV near the bottom of the slide. Therefore, focusing on tissue at the center of the slide is recommended for optimal image quality.

Write down all numbers in log sheet.


When done, put SED back into standby mode so you don’t ablate your tissue sample.
Acquiring Data & Monitoring
Acquiring Data & Monitoring
Load back FOVs from exported json file.


Start Run.
  • Observe first few points in GUI for channel dropouts, low counts, any focus issues.
Look at your images in the Ionpath MIBIViewer.



Post-Run
Post-Run
Set the machine to STANDBY mode in MIBI Control.