TITLE: Tile/SED/Array Interface (TSAI) for Multiplexed Ion Beam Imaging (MIBI) run setup
AUTHORS: Christine Camacho, Hadeesha Piyadasa, Benjamin Oberlton, Alex Kong, Cameron Sowers, Sricharan Reddy Varra, Albert Tsai

[DESCRIPTION]
The Multiplexed Ion Beam Imager (MIBI) is a next-generation mass spectrometry-based microscope which generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in its operation occurs when users select the physical locations on the tissue for imaging. As the scale and complexity of MIBI experiments have increased, the manufacturer-provided interface and third party tools have become increasingly unwieldy for imaging large tissue microarrays and tiled tissue areas. Thus, we have developed a web-based graphical interface layer for users to set imaging locations using intuitive mouse gestures such as drag-and-drop, click-and-drag, and polygon draw. Written according to web standards already built into modern web browsers, it requires no installation of external programs, extensions, or patches. Of interest to the hundreds of current MIBI users, our interface simplifies setup of complex experiments and has drastically reduced our setup time.

[STEPS]
SECTION: Load the MIBI slide and and create a template .json file
4. Log into MIBItracker in the internet browser.
SECTION: MIBIscope general guidelines
1. Keep the MIBIscope in "Standby" mode when not actively setting up a run. Keeping it in SED mode ablates (burns through) tissue as you navigate, so work quickly and efficiently.
SECTION: MIBIscope general guidelines
2. Perform run setup (including FOV navigation) in "QC - 300μm" mode.
SECTION: MIBIscope general guidelines
3. Perform detector calibration before imaging runs, at most once per day.
SECTION: Load the MIBI slide and and create a template .json file
5. In the “Slides” tab, accession a new slide and add a new section.
SECTION: Load the MIBI slide and and create a template .json file
6. In the “Resources” tab, select or create a marker panel.
SECTION: Load the MIBI slide and and create a template .json file
7. In the “Sections” tab, add the new section to the panel.
SECTION: Load the MIBI slide and and create a template .json file
8. Log into MIBIcontrol in the internet browser.
SECTION: Load the MIBI slide and and create a template .json file
9. Load the MIBI slide by clicking “Exchange Sample” and selecting the new slide.
SECTION: Load the MIBI slide and and create a template .json file
10. Create a template FOV by clicking “Add FOV” and setting the frame dimensions, FOV size, dwell time, imaging mode, and section ID.
SECTION: Load the MIBI slide and and create a template .json file
11. Export (download) the FOV list to a .json file.
SECTION: Load the MIBI slide and and create a template .json file
12. Download the optical image to a .png file.
SECTION: Optical image-stage motor coregistration
13. Open the TSAI web UI in the internet browser.
SECTION: Optical image-stage motor coregistration
14. If coregistration has not been previously performed, the optical coregistration menu should automatically open. If it has been performed and is adequate, these steps do not need to be repeated.
SECTION: Optical image-stage motor coregistration
15. Open the “Optical Coregistration” menu.
SECTION: Optical image-stage motor coregistration
16. Click “Copy automatic registration code to clipboard”.
SECTION: Optical image-stage motor coregistration
17. Open MIBIcontrol in the internet browser.
SECTION: Optical image-stage motor coregistration
18. Type “Ctrl+Shift+J” to open the browser console, or right click on the page and click “Inspect”, then open the “Console” tab.
SECTION: Optical image-stage motor coregistration
19. Paste the code into the console and press “Enter”.
SECTION: Tiled SED scan
21. Load the .png and .json files by dragging and dropping them onto the TSAI web UI.
SECTION: Tiled SED scan
22. Open the “SED Tiler” menu and click on a text box in the top row.
SECTION: Tiled SED scan
23. Click (± drag) on the optical image to select the top left corner for the SED scan.
SECTION: Tiled SED scan
24. Press the “D” key or click on a text box in the second row in the “SED Tiler” menu.
SECTION: Tiled SED scan
25. Click (± drag) on the optical image to select the bottom right corner for the SED scan.
SECTION: Tiled SED scan
26. In the “SED Tiler” menu click “Copy SED scan and shift correction code to clipboard”.
SECTION: Tiled SED scan
27. Open MIBIcontrol in the internet browser.
SECTION: Tiled SED scan
28. Paste the code into the console and press “Enter”.
SECTION: Tiled SED scan
29. Put the MIBI into SED mode on the “QC - 300μm” setting, move to an area you will not be acquiring in your run, and adjust the gain, focus, and stigmation.
SECTION: Tiled SED scan
30. Type “Shift+T” to start the tiled SED scan.
SECTION: Tiled SED scan
31. When finished, it should automatically save a new .png file of the tiled SED image. You may add characters to the beginning of the file name but do not modify any other part of the file name.
SECTION: Tiled SED scan
32. If the tiles in the SED are misaligned, contact Ionpath support to adjust the motor and imaging beam, or you may attempt manual software correction using the other keyboard controls.
SECTION: Tiled SED scan
33. If the tiled SED is adequate, press “Escape”.
SECTION: Tiled SED scan
34. Return to the TSAI web UI.
SECTION: Tiled SED scan
35. Drag and drop the SED .png file onto the TSAI web UI.
SECTION: Tiled SED scan
36. Click on the “SED” tab and adjust the zoom.
SECTION: Tiled SED scan
37. To adjust image brightness and contrast and/or drawing options such as line thickness and cursor size, use the “Slide Options” menu above the SED image.
SECTION: Optical image-stage motor coregistration
20. Click the link generated in the console. This will load the coregistration into the TSAI web UI and save it as a cookie, so it persists and does not need to be repeated unless there is a change to the instrument hardware.
SECTION: Tissue microarray (TMA)
38. If setting FOVs for a grid of TMA spots, set the pattern of FOVs to be replicated by adjusting the columns and rows in the relevant tile of the “Tiles” column and checking/unchecking the boxes in the “Map”.
SECTION: Tissue microarray (TMA)
39. Click “TMA” to open the TMA options menu. Set the number of rows and columns in the TMA. If necessary, add a naming prefix and edit the starting row and column numbering.
SECTION: Tissue microarray (TMA)
40. On the SED image, click on the four corners of the TMA. Click and drag the circled corners to adjust the positioning of the crosshairs so they best match the TMA spots.
SECTION: Tissue microarray (TMA)
41. Click “Build TMA” from the TMA options menu.
SECTION: Tissue microarray (TMA)
42. Hover over each tile in the “Tiles” column to check its positioning. To adjust, click “Move”. Then click and drag on the slide image or press the keyboard arrow keys.
Holding the “Shift” key and pressing arrow keys will move a farther distance.
Holding the “Alt” (Windows) or “Opt” (Mac) key and pressing arrow keys will move a shorter distance.
When “Move” is selected, pressing “A” will go to the previous tile and pressing “D” will go to the next tile.
To remove the tile, uncheck the checkbox next to the tile name or click “Delete”.
To adjust other tile settings, click the “=” button to expand the settings menu if it is not visible.
SECTION: Area/polygon tile
43. If setting FOVs to cover a contiguous area of tissue, click “Polygon” in the relevant tile of the “Tiles” column.
SECTION: Area/polygon tile
44. Click on the SED image to set the vertices/corners of the area to be tiled. Double-click to close the polygon and cover the area with FOVs.
SECTION: Area/polygon tile
45. Scroll to the bottom of the “Tiles” column and click the “=” button in the new polygon tile to see the tile map.
SECTION: Area/polygon tile
46. Toggle individual tiles on or off by clicking on the tile map, or by clicking “Clicker” and clicking on the tiled FOVs in the SED image.
SECTION: Area/polygon tile
47. To toggle off multiple FOVs, click on “Eraser” and then click and drag on the tiled FOVs in the SED image.
SECTION: Area/polygon tile
48. To toggle on multiple FOVs, click on “Clicker” and then click and drag on the empty areas in the SED image covered by the tile map. You may need to increase the numbers of rows or columns, insert rows above using the “▲” button, or insert columns to the left using the “◄” button.
SECTION: Area/polygon tile
49. To adjust tile positioning, click “Move”. Then click and drag on the slide image or press the keyboard arrow keys.
Holding the “Shift” key and pressing arrow keys will move a farther distance.
Holding the “Alt” (Windows) or “Opt” (Mac) key and pressing arrow keys will move a shorter distance.
SECTION: FOV navigation and adjustment
50. If SED tiling is misaligned, you may adjust exact FOV positioning in the MIBIcontrol SED mode with the aid of the below keyboard controls.
SECTION: FOV navigation and adjustment
51. Open the “FOV Navigation/Adjustment” menu below the SED image.
SECTION: FOV navigation and adjustment
52. Click “Copy FOV navigation code to clipboard”.
SECTION: FOV navigation and adjustment
53. Open MIBIcontrol in the internet browser.
SECTION: FOV navigation and adjustment
54. Put the MIBI into SED mode and adjust the gain, focus, and stigmation.
SECTION: FOV navigation and adjustment
55. Type “Ctrl+Shift+J” to open the browser console, or right click on the page and click “Inspect”, then open the “Console” tab.
SECTION: FOV navigation and adjustment
56. Paste the code into the console and press “Enter”.
SECTION: FOV navigation and adjustment
57. The code will automatically navigate to the first FOV and the exact FOV positioning displayed in the MIBIcontrol SED image.
SECTION: FOV navigation and adjustment
58. To adjust the SED magnification, press the “B” (50 μm), “N” (100 μm), “M”, (200 μm), “,” (400 μm), “.” (800 μm), or “/” (maximum) keys. The 400 μm, 800 μm, and maximum settings will also draw corners indicating the FOV for smaller magnifications.
SECTION: FOV navigation and adjustment
59. To move the FOV, press the keyboard arrow keys. Save the position by pressing “W”.
Holding the “Shift” key and pressing arrow keys will move a farther distance.
Holding the “Alt” (Windows) or “Opt” (Mac) key and pressing arrow keys will move a shorter distance.
Note that only R1C1 of any given tile can be moved.
SECTION: FOV navigation and adjustment
60. To toggle an FOV on or off, press “T”.
SECTION: FOV navigation and adjustment
61. To change the FOV size, press “2” (200 μm), “4” (400 μm), or “8” (800 μm). The raster dimensions will be scaled proportionately.
SECTION: FOV navigation and adjustment
62. To save an image of the SED image and overlaid crosshair, press “S”.
SECTION: FOV navigation and adjustment
63. To save a draft of the adjustments to a .txt file, press “X”.
SECTION: FOV navigation and adjustment
64. When satisfied, press “D” to go to the next FOV, or “A” to go back to the previous FOV.
SECTION: FOV navigation and adjustment
65. Repeat steps 59-64 for all FOVs.
SECTION: FOV navigation and adjustment
66. When finished with all FOVs, press “X” or “Escape”, which will automatically save adjustments to a .txt file and attempt to copy them to the clipboard.
SECTION: FOV navigation and adjustment
67. Return to the TSAI web UI.
SECTION: FOV navigation and adjustment
68. Drag and drop the .txt file onto the TSAI web UI or paste the adjustments into the text box in the “FOV Navigation/Adjustment” menu.
SECTION: FOV navigation and adjustment
69. Click “Adjust” to apply adjustments to the tiles in the “Tiles” column.
SECTION: JSON file generation and import
70. Check the list of tiles and estimated run time under “Output” below the “Tiles” column.
SECTION: JSON file generation and import
71. To create a JSON file in the same order as the FOVs in the “Tiles” column, click “Create JSON file (sequential order)”.
SECTION: JSON file generation and import
72. To create a JSON file in random order, click one of the “Create JSON file (random order” buttons. 
“Different tiles mixed together” randomizes in a way that randomly jumps between different tiles. 
“Keeping tiles together” randomizes the order of tiles and within each tile, but only jumps to a new tile when the prior one is complete.
SECTION: JSON file generation and import
73. Save the resulting .json file to an appropriate location on the computer.
SECTION: JSON file generation and import
74. Return to MIBIcontrol.
SECTION: JSON file generation and import
75. Click “Import FOVs” and select the generated .json file.
SECTION: Focusing and run start
76. For final focusing, move to an area that you will not be acquiring in your run, in order to avoid ablating areas that you will be acquiring.
SECTION: Focusing and run start
78. Check the current and adjust lens 1 as necessary.
SECTION: Focusing and run start
79. Click “Start run”.
SECTION: Focusing and run start
77. Focus in your acquisition mode.
SECTION: Focusing and run start
78.1. In the "Target Selection" drop-down menu, select "Faraday Cup".
SECTION: Focusing and run start
78.2. Set the mode to "Spot".
SECTION: Focusing and run start
78.3. Set the lens 1 voltage such that current falls within the range indicated in the MIBIscope Quick Start Guide, e.g. 9.5 ± 0.2 nA for Coarse. Increasing lens 1 decreases the current. Decreasing lens 1 increases the current.
SECTION: Focusing and run start
77.1. Set the imaging mode to that you will be using for acquisition (e.g., Coarse, Fine, Superfine).
SECTION: Focusing and run start
77.2. Set lens 2 such that the center of the SED is in sharp focus, and the top and the bottom are equally blurred.
SECTION: Focusing and run start
77.3. Set the stigmation angle and magnitude to correct residual blurring.