Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope using the Tile/SED/Array Interface

Abstract

Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique that generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in 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, a web-based, interactive, what-you-see-is-what-you-get (WYSIWYG) graphical interface layer - the tile/SED/array Interface (TSAI) - was developed for users to set imaging locations using familiar and intuitive mouse gestures such as drag-and-drop, click-and-drag, and polygon drawing. Written according to web standards already built into modern web browsers, it requires no installation of external programs, extensions, or compilers. Of interest to the hundreds of current MIBI users, this interface dramatically simplifies and accelerates the setup of large, complex MIBI runs.

Introduction

Multiplexed ion beam imaging (MIBI) is a technique to image 40+ proteins simultaneously on histologic tissue sections at up to 250 nm resolution^1,2,3. After a histologic tissue section is stained using antibodies tagged with isotopically pure elemental metals, the MIBI instrument performs secondary ion mass spectrometry to simultaneously quantify all the isotopes - and thus expression of all 40+ antigens - at individual spots on the tissue. Performed across grids of millions of spots, the resulting 40+ plex images of protein expression enable the delineation of cell boundaries and identification of specific cell types while preserving spatial context^1,2,3,4. This technique has been used by hundreds of users at roughly 20 sites to study the cellular composition, metabolic profiles, and/or architecture of dozens of tissue types as part of examining the immune response to tumors, tissue inflammation caused by infectious agents, neuropathology of dementia, and immune tolerance in pregnancy^{5,6,7,8,9,10,11}.

A key bottleneck in MIBI instrument operation is setting up fields of view (FOVs) - 200 x 200 µm² to 800 x 800 µm² areas of the tissue - for imaging. The MIBI images one FOV at a time, up to 800 x 800 µm², thus imaging larger areas requires stitching multiple FOVs together. Imaging a tissue microarray (e.g., eight circular tissues in Figure 1A) involves placing multiple FOVs spaced apart. To set up FOVs, the manufacturer interface provides 1) an optical camera image of the slide with a crosshair that roughly corresponds to the specified imaging coordinate (Figure 1A) and 2) a secondary electron detector (SED) image that shows the exact area at the coordinate, reportedly accurate to within 0.1 µm (Figure 1B). First, the user roughly positions a single FOV using the optical image. Because the image resolution is only about 60 µm per pixel, if the placement is off by two pixels (2 pixels x 60 µm per pixel), a standard 400 µm FOV will be off by 30%. Thus, the user must use the SED image to fine-tune the position - a tedious sequence of a dozen steps involving multiple popup windows, typing coordinates into text boxes, slowly nudging the SED with directional control buttons, and often even writing down coordinates on paper (Supplementary Figure 1). This process must be repeated for each spot of a 100+ core tissue microarray (TMA). Some third-party tools can help with the initial rough positioning¹². However, they still require some programming knowledge, and final positioning is still done through the dozen-step process. It is also highly troublesome to position grids of adjacent FOVs, which will be later stitched together into a tiled panoramic image.

Thus, the tile/SED/array Interface (TSAI) was developed with the goal of enabling users to rapidly position large numbers of FOVs using an intuitive, interactive graphical interface. TSAI consists of two main components: 1) A web-based graphical user interface (web UI) for rapidly placing TMA points and tissue tiles, and 2) Integrations into the MIBI user control interface for generating a tiled SED image and adjusting FOV positions. If only using the optical image, many FOVs can be roughly positioned and then quickly adjusted using the FOV navigation/adjustment tools (Figure 2, TSAI, left branch). However, if the SED tiling is performed, FOVs can be accurately positioned on the tiled SED image without needing further adjustments in SED mode (Figure 2, TSAI, right branch). Of general interest to hundreds of current MIBI users, these tools make tiling and TMA positioning very simple even for novices and reduce complex MIBI run setups from several hours to a few dozen minutes.

Protocol

1. Loading of TSAI

1. Run TSAI by opening https://tsai.stanford.edu/research/mibi_tsai in the web browser of the MIBI user control computer.
   1. This instance of TSAI contains custom presets which do not apply to all instruments. When using it, build tiles only from template FOV(s) as generated below in step 2.6. TSAI runs locally within the web browser, and no image, .json, or file name data is sent to or stored on the server.
2. Alternatively, set up TSAI on any website with custom presets for any instrument.
   1. Go to https://github.com/ag-tsai/mibi_tsai and download the mibi_tsai_standalone directory. Alternatively, download the Supplementary Coding File 1 .zip file and unzip the contents to a directory titled mibi_tsai_standalone.
   2. Open mibi_tsai_standalone/_resources/index.js in any text editor.
   3. If necessary, edit the FOV size, dwell time/timing choice, raster size, FOV JSON, and recommended preset settings in index.js to match the instrument's settings. This mainly applies to customized instruments, but dwell time/timing choice pairs should be checked regardless. Save index.js.
   4. Upload mibi_tsai_standalone to any web server accessible through the internet, e.g., a lab web site or university-hosted web site.
   5. Open mibi_tsai_standalone/index.html in the web browser of the MIBI user control computer.

2. Loading the MIBI slide and creating a template file

1. Log into the MIBI experiment tracker (manufacturer-provided web interface for managing scan-related metadata) in the web browser.
2. In the Slides tab, add a new slide and add a new section (Supplementary Figure 2A-B). In the Resources tab, select or create a marker panel (Supplementary Figure 2C).
3. In the Sections tab, add the new section to the panel (Supplementary Figure 2D).
4. Log into the MIBI user control interface in the web browser. Load the MIBI slide by clicking Exchange Sample and selecting the new slide (Supplementary Figure 3A).
5. Create a template FOV by clicking Add FOV (Supplementary Figure 3B) and setting the frame dimensions, FOV size, dwell time, imaging mode, and section ID.
6. Export (download) the FOV list to a .json file (Supplementary Figure 3C). Download the optical image as a .png file (Supplementary Figure 3D).

3. Optical image-stage motor coregistration

1. Open the TSAI web UI in the web browser. If coregistration has not been previously performed, the optical coregistration menu should open automatically. If it has been performed and is adequate, do not repeat these steps.
2. Open the Optical Coregistration menu. Click Copy Automatic Coregistration Code to Clipboard (Supplementary Figure 4A).
3. Open the MIBI user control interface in the web browser. Press Ctrl+Shift+J to open the browser console, or right-click on the page and click Inspect, then open the Console tab (Supplementary Figure 4B).
4. Paste the code into the console and press Enter. Click the link generated in the console (Supplementary Figure 4C). 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.

4. Tiled SED scan

1. Load the optical image .png and .json files from step 2.6 by dragging and dropping them onto the TSAI web UI.
2. Open the SED Tiler menu and click on a text box in the top row (Supplementary Figure 5A).
3. Click (± drag) on the optical image to select the top left corner for the SED scan (Supplementary Figure 5B).
4. Press the D key or click on a text box in the second row in the SED Tiler menu.
5. Click (±​ drag) on the optical image to select the bottom right corner for the SED scan.
6. In the SED Tiler menu, click Copy SED Scan and Shift Correction Code to Clipboard (Supplementary Figure 5C).
7. Open the MIBI user control interface in the web browser. Paste the code into the console and press Enter (Supplementary Figure 5D).
8. Put the MIBI into SED mode on the QC - 300 µm setting, move to an area that will not be acquired, and adjust the gain, focus, and stigmation.
   1. Adjust the brightness and contrast of the SED image without changing the gain. Press B to increase the brightness or Shift+B to decrease it. Press C to increase the contrast or Shift+C to decrease it. Press Shift+V to reset both the brightness and contrast.
9. Press Shift+T to start the tiled SED scan.
10. When finished, it should automatically save a new .png file of the tiled SED image (Figure 3). Characters may be added to the beginning of the file name but do not modify any other part of the file name.
11. If specific tiles are out of focus or otherwise improperly scanned, rescan them.
    1. Press Shift+R to add a tile to the rescan queue. A dialog box will open, prompting the user for the row and column of the tile. The numbers are zero-indexed, thus entering 8,0 queues the ninth row, the first column.
    2. After adding all the relevant tiles to the queue, press Shift+T to rescan. When finished, it should automatically save a new .png file of the tiled SED image.
12. Critical step: Inspect the tiled SED scan for large misalignments (Figure 3C-D). If present, contact manufacturer support to adjust the motor and imaging beam or attempt manual software correction using the keyboard controls in steps 4.12.1 to 4.12.9 (Supplementary Figure 6A).
    1. To check SED-stage motor alignment, move to an area of the slide without tissue. Press Shift+5 to burn five 400 µm FOVs in a checkerboard pattern (Supplementary Figure 6B-C) or Shift+9 to burn a 3 x 3 pattern of 400 µm FOVs (Supplementary Figure 6D-E).
    2. If the FOV columns are too far apart, press 1 and set the x f(x) value to a negative decimal, typically between -0.0025 and -0.1.
    3. If the third-row FOVs are shifted leftward relative to the first-row FOVs, press 2 and set the x f(y) value to a positive decimal, typically between 0.0025 and 0.1.
    4. If the third column FOVs are shifted downward relative to the first column FOVs, press 3 and set the y f(x) value to a negative decimal, typically between -0.0025 and -0.1.
    5. If the FOV rows are too far apart, press 4 and set the y f(y) value to a negative decimal, typically between -0.0025 and -0.1.
    6. Iteratively repeat steps 4.12.1 to 4.12.5 until the checkerboard and 3 x 3 patterns form a roughly straight grid (Supplementary Figure 6C, E).
    7. Press S to save a .png image of the pattern with the correction values in the file name.
    8. Drag and drop this .png file onto the TSAI web UI to load the values and save them to the browser cookie.
    9. Perform tiled SED scans to check the coefficients. Based upon the same principles as in steps 4.12.2 to 4.12.5, make further adjustments to the coefficients to correct any misalignments in the tiled SED images.
13. If the tiled SED is adequate, press Escape. Return to the TSAI web UI.
14. Drag and drop the tiled SED .png file onto the TSAI web UI (Supplementary Figure 5E).
15. Click on the SED tab and adjust the zoom (Supplementary Figure 5F).
16. 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.
17. Keyboard shortcuts are available and most shown next to the image controls: Press Z to zoom in and Shift+Z to zoom out. Press B to increase brightness or Shift+B to decrease it. Press C to increase contrast or Shift+C to decrease it. Press Shift+V to reset both the brightness and contrast. Press L to toggle labels above the tiles. Press O to toggle 5 mm radius circles drawn around focus sites.

5. Tissue microarray (TMA)

1. If setting FOVs for a grid of TMA spots, first set the pattern of FOVs to be replicated. In the relevant tile of the Tiles column, adjust the columns and rows (Figure 4A) and check/uncheck the boxes in the map (Figure 4B), as well as adjusting other FOV settings as necessary.
2. In the relevant tile, click TMA to open the TMA options menu (Figure 4C). Set the number of rows and columns of TMA spots (Figure 4D). If necessary, add a naming prefix (Figure 4E) and edit the starting row and column numbering (Figure 4F).
3. On the slide image, click on the four corners of the TMA (Figure 4G-J). Click and drag the circled corners to adjust the positioning of the crosshairs so they best match the TMA spots.
4. Click Build TMA from the TMA options menu (Figure 4K).
5. Hover over each tile in the tiles column to check its positioning. To adjust, click Move (Figure 4L). Then click and drag on the slide image or press the keyboard arrow keys.
   1. Hold the Shift key while pressing arrow keys to move a farther distance. Hold the Alt (Windows) or Opt (Mac) key while pressing arrow keys to move a shorter distance.
   2. When move is selected, press T to uncheck the checkbox next to the tile name, remove it from view, and omit it from any subsequently generated .json files. Alternatively, uncheck the checkbox directly with the mouse (Figure 4M) or remove it entirely by clicking Delete.
   3. When move is selected, press 2, 4, or 8 to set the FOV size to 200 µm, 400 µm, or 800 µm, respectively, and the raster dimensions will be scaled proportionately such that the imaging resolution remains unchanged.
   4. When move is selected, press A to go to the previous tile or press D to go to the next tile.
   5. To adjust other tile settings, click the ≡ button to expand the settings menu if it is not visible.

6. Area/polygon tile

1. If setting FOVs to cover a contiguous area of tissue, first adjust FOV settings as necessary in the relevant tile of the Tiles column.
2. In the relevant tile, click Polygon (Figure 5A). Click on the slide image to set the vertices/corners of the area to be tiled (Figure 5B-C). Double-click to close the polygon and cover the area with FOVs (Figure 5D).
3. Scroll to the bottom of the Tiles column and click the ≡ button (^ when expanded, Figure 5E) in the new polygon tile to see the tile map.
4. Toggle individual tiles ON or OFF by clicking on the tile map (Figure 5F), or by clicking Clicker (Figure 5G) and clicking on the tiled FOVs in the slide image.
5. To toggle off multiple FOVs, click on Eraser and then click and drag on the tiled FOVs in the slide image (Figure 5H).
6. To toggle on multiple FOVs, click on Clicker (Figure 5G) and then click and drag on the empty areas in the slide image covered by the tile map.
7. To insert the rows above, click the ▲ button (Figure 5I). To insert columns to the left, click the ◄ button (Figure 5J).
8. To adjust tile positioning, click Move (Figure 5K). Then click and drag on the slide image, press the keyboard Arrow keys, or use other controls described in steps 5.5.1 to 5.5.5.

7. FOV navigation and adjustment

1. If SED tiling is misaligned or the optical image crosshair does not reflect the actual stage motor position, adjust FOV positions in SED mode in the MIBI user control interface with the aid of the below keyboard controls.
2. Open the FOV navigation/adjustment menu below the slide (optical or SED) image. Click Copy FOV Navigation Code to Clipboard.
3. Open the MIBI user control interface in the web browser. Put the MIBI into SED mode and adjust the gain, focus, and stigmation.
4. Press Ctrl+Shift+J to open the browser console, or right-click on the page and click Inspect, then open the Console tab.
5. Paste the code into the console and press Enter. The code will automatically navigate to the first FOV and the exact FOV positioning displayed in the SED image of the MIBI user control interface.
6. Adjust the brightness and contrast of the SED image without changing the gain. Press B to increase the brightness or Shift+B to decrease it. Press C to increase the contrast or Shift+C to decrease it. Press Shift+V to reset both the brightness and contrast.
7. To adjust the SED magnification, press M (200 µm), , (400 µm), . (800 µm), or / (maximum) keys.
8. To move the FOV, press the keyboard Arrow keys. Save the position by pressing W. Hold the Shift key while pressing arrow keys to move a farther distance. Hold the Alt (Windows) or Opt (Mac) key while pressing arrow keys to move a shorter distance. Note that only R1C1 of any given tile can be moved.
9. To toggle an FOV ON or OFF, press T. To change the FOV size, press 2 (200 µm), 4 (400 µm), or 8 (800 µm). The raster dimensions will be scaled proportionately such that the imaging resolution remains the same.
10. To save an image file of the SED image and overlaid crosshair, press S. To save a draft of the adjustments to a .txt file, press X.
11. When satisfied, press D to go to the next FOV, or A to go back to the previous FOV. Repeat steps 7.6 to 7.11 for all FOVs.
12. When finished with all FOVs, press X or Escape. Adjustments will be saved to a .txt file and copied to the clipboard.
13. Return to the TSAI web UI. 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.
14. Click Adjust to apply adjustments to the tiles in the Tiles column.

8. JSON file generation and import

1. Below the Tiles column, under Output, check the list of tiles and estimated run time (Supplementary Figure 7A).
2. Under Group, select an option for FOV grouping (Supplementary Figure 7B). Grouping has no effect on the sequentially ordered .json file.
   1. For the randomized .json file, grouping FOVs by tile will order FOVs such that all the FOVs within a given tile stay together, even though the tiles are in random order.
   2. For the randomized .json file, do not group FOVs will randomly order FOVs such that FOVs from different tiles are intermixed.
   3. If in-run autofocus has been specified, FOVs will automatically be grouped by the closest autofocus site.
3. Under split, select an option for splitting into multiple .json files (Supplementary Figure 7C).
   1. Do not split will keep all FOVs in only one .json file.
   2. Split by every # FOVs will split FOVs across multiple .json files, where each file contains the specified number of FOVs.
   3. Split by every # hours # minutes will split FOVs across multiple .json files, where each file's estimated run time is roughly the specified amount of time.
4. View and rearrange the order of FOVs in the .json files by opening the Rearrange menu (Supplementary Figure 7D). To move an FOV, click and drag it to the desired position. The other FOVs will interactively rearrange around the dragged FOV.
5. To save the .json file(s), click on the FOVs button(s) beneath the rearrange menu. The sequential .json puts FOVs in order by tile, then row, then column (Supplementary Figure 7E). The random .json randomizes FOVs within the groups as selected in step 8.2 (Supplementary Figure 7F).
6. To save an image of the tissue with the FOVs and applied display options (tile labels, brightness, contrast, etc.), click Save Tiled Image (Supplementary Figure 7G). This is often useful for record-keeping and sharing with collaborators.
7. Return to the MIBI user control interface. Click Import FOVs and select the generated .json file. Adjust the focus, stigmation, and current as necessary and click Start Run.

Representative Results

TSAI provides two methods for setting up FOVs (Figure 2). One uses only the optical image (Figure 2, TSAI, left branch), similar to other existing methods. The second method - generating a tiled SED image - is unique to TSAI (Figure 2, TSAI, right branch). TSAI draws FOVs accurately onto this image, eliminating the need to spend hours nudging FOVs into place in the manufacturer interface SED mode. However, the correction coefficients for SED tiling must be set properly, or else the resulting FOV positions may not be accurate.

SED tiling coefficients can be assessed by inspecting the tiled SED scan of a large tissue section, at least 1.5 cm in size (steps 4.1 to 4.11). The expected result of a proper setup is an image with minimal misalignments in the tissue between adjacent tiles (Figure 3A-B). If the SED imaging square is at an angle relative to the stage motor, or if there is step motor miscalibration (e.g., a command to move 400 µm moves 390 µm in reality), then the tiled SED scan will show significant misalignments, duplicated areas, and/or gaps (Figure 3C-D). Ideally, these motor inaccuracies would be corrected by the manufacturer such that all the coefficients could be set to 0. Otherwise, coarse manual corrections may be performed using patterned slide burns (steps 4.12.1 to 4.12.8, Supplementary Figure 6) before finer corrections using large, tiled SED scans (step 4.12.9).

To further check positioning accuracy, one may verify that the FOV squares drawn on the tiled SED image match those shown in the manufacturer interface SED mode after navigating to the FOV coordinate (Supplementary Figure 1A-B). One may also use the FOV navigation/adjustment tools (step 7) to quickly perform these checks for many FOVs.

Figure 1: Current MIBI user control interface. MIBI user control interface showing a representative slide with tissue sections of the brain (upper two tissues) and a small tissue microarray (TMA, lower eight circular tissues). (A) The optical camera image of the entire slide. The yellow crosshair (X) denotes the current location of the SED image. (B) The secondary electron detector (SED) image of the location marked by the yellow crosshair. Each side of the imaged square is 1400 µm. (C) The import fields of view (FOVs) feature. Please click here to view a larger version of this figure.

Figure 2: Schematic of Tile/SED/array interface (TSAI) workflow compared to standard FOV setup. First, the slide is loaded into the MIBI. Standard FOV setup involves positioning FOVs one at a time. TSAI proceeds with optical image-stage motor coregistration (step 3) and loading the optical image into TSAI. One branch of TSAI (left branch) uses only the optical slide image to roughly position FOVs (steps 5 and 6), followed by fine adjustment using FOV navigation/adjustment tools (step 7). The right branch generates a tiled SED scan (step 4). FOVs are accurately positioned on the tiled SED image (steps 5 and 6) without the need for further adjustment. After creating the .json file and importing it into the MIBI user control interface (step 8), the MIBI operation proceeds per protocol. The tiled SED scan shows the scale at the bottom right of the image. Please click here to view a larger version of this figure.

Figure 3. Representative tiled SED scans of tonsil sections. (A) A properly tiled SED image shows minimal misalignments between SED tiles. (B) The dotted area is enlarged. (C) Suboptimal setup results in large misalignments between SED tiles. (D) The dotted area is enlarged with arrows pointing to discontinuities. Scales are shown at the bottom right of each image. Please click here to view a larger version of this figure.

Figure 4. Interactive graphical interface for positioning tiles/FOVs on a tissue microarray (TMA). At the left, a tiled SED image of a TMA is loaded into the TSAI web UI, with the scale bar shown at the upper left. On the right is the tiles column containing the tile/FOV options and tools. As described in step 5, (A, B) users first set the pattern of FOVs to copy, (C-F) then set the dimensions and naming options. (G-J) After clicking and adjusting the corners and (K) building the tiles, (L) move the tiles into place or (M) remove tiles when there is no tissue. Please click here to view a larger version of this figure.

Figure 5. Interactive graphical interface for building a tile with FOVs covering a polygonal area. At the left, a tiled SED image of a tonsil is loaded into the TSAI web UI, with the scale bars shown at the upper left. At the right is the Tiles column containing the tile/FOV options and tools. As described in step 6, (A) users select the polygon tool, (B-C) then click to set the vertices, (D) and double click to close the polygon. (E) Expanding the menu shows additional options. (F) The FOV map can be edited by clicking the checkboxes. FOVs can be individually toggled on and off by clicking on the slide image, (G) toggled on by click and drag , (H) or erased by click and drag. Rows may be inserted (I) above or (J) columns inserted to the left. (K) The entire tile can be moved by click and drag. Please click here to view a larger version of this figure.

Supplementary Figure 1. Current workflow for setting up FOVs using the manufacturer's user control interface. Using the optical image panel at the left, the user roughly positions a single FOV. Then, they fine-tune its position through a sequence of 12-13 steps: 1. Type the FOV X coordinate into the stage position X coordinate text box. 2. Type the FOV Y coordinate into the stage position Y coordinate text box. 3. Click Move. 4. Click Jog Stage. 5. Click the Arrow buttons in the jog stage popup window to adjust the stage position until the SED image shows the desired area for imaging. 6. Optionally, use a screenshot tool to capture the SED image and crosshair overlay. 7. Write the new adjusted X coordinate on a piece of paper. 8. Write the new adjusted Y coordinate on a piece of paper. 9. Click on the Pencil/Paper icon to edit the FOV coordinate. 10. Type the coordinate from step 8 into the FOV center point X text box. 11. Type the coordinate from step 9 into the FOV center point Y text box. 12. Select the appropriate section from the drop-down menu. 13. Click Confirm. The position for that single FOV is now adjusted. This sequence is repeated for every FOV, with some slides exceeding 100 FOVs. Please click here to download this File.

Supplementary Figure 2. Setting up a slide, section, and panel in the MIBI experiment tracker, per usual MIBI operation protocol. (A-B) Before a slide can be loaded into the MIBI, users must use the Project Data/Slides tab to create a slide that contains a section. (C-D) Then, use the Resources/Panels tab to apply a panel to the section Please click here to download this File.

Supplementary Figure 3. Loading a slide, creating a template .json, and downloading the optical image in the MIBI user control interface. (A) As described in step 1, users perform a sample exchange to load the slide into the MIBI. (B) Then they create a template FOV, (C) save the. json and (D) save the optical image for import into the TSAI web UI. Each side of the SED image square is 1400 µm. Please click here to download this File.

Supplementary Figure 4. Performing optical image-stage motor coregistration. As described in step 2, users: 1. Open then optical coregistration menu; 2. Click the button to copy the code to the clipboard; 3. Open the MIBI user control interface JavaScript console; 4. Paste the code into the console and press Enter; and 5. Click on the resulting link. Please click here to download this File.

Supplementary Figure 5. Performing a tiled SED scan. A representative TMA slide is shown in the TSAI web UI. As described in step 3, (A-B) users first select corners of the rectangular area to scan. (C) Then click the button to copy the code to the clipboard. (D) After running the code in the JavaScript console and setting scan parameters (particularly gain and focus), they press Shift+T to begin the automatic scan. In (D), each side of the SED image square is 1639 µm. (E) After the tiled SED image has been generated, it is loaded into the TSAI web UI. (F) Slide display options such as zoom and brightness can then be set. In (F), the scale of the tiled SED image is shown at the upper left of the image. Please click here to download this File.

Supplementary Figure 6. Manual correction of SED misalignments. (A) A schematic of a five-square checkerboard burn, before (left) and after (right) adjustment of the correction coefficients as described in step 4.13. The x f(x) and y f(y) coefficients are corrections for the step motor being slightly miscalibrated in the x and y axis, respectively. The y f(x) coefficient is an angular correction where a movement in the x direction requires a correction in y. The x f(y) coefficient is an angular correction where a movement in the y direction requires a correction in x. (B) An actual five-square checkerboard burn, before (left) and after (right) adjustment of the coefficients. (C) An actual nine-square checkerboard burn, before (left) and after (right) adjustment of the correction coefficients. SED images shown are all 1300 µm on each side. Please click here to download this File.

Supplementary Figure 7. Generating the .json file and importing it into the MIBI user control interface. As described in step 7, (A) one should check the FOVs and estimated time before generating the .json file. (B) After selecting a grouping option and (C) a splitting option, (D) the user may change the order of the FOVs in the .json file(s). (E) By default, the Sequential .json orders FOVs just as they are ordered in the TSAI web UI - by tile, then row, then column. (F) The Random .json randomizes the FOVs within the groups specified in (B). (G) An image of the slide and tiles is useful for record-keeping and sharing with collaborators. (H) The .json is then loaded into the MIBI user control interface before starting the run. Please click here to download this File.

Supplementary Coding File 1: .zip file containing TSAI code. GitHub repository maintained at https://github.com/ag-tsai/mibi_tsai. Please click here to download this File.

Discussion

Multiplexed ion beam imaging (MIBI) is a powerful technique for dissecting detailed cellular phenotypes and tissue histoarchitecture^{5,6,7,8,9,10,11}. Computational efforts around MIBI have largely focused on processing the data after imaging, but little has been done to improve the instrument software's usability for common applications such as large TMAs and tiled tissue areas. As the scale and complexity of MIBI experiments have increased, the manufacturer-provided instrument interface and third-party tools have become increasingly unwieldy for these tasks¹². Thus, the tile/SED/array interface (TSAI) was developed to enable users to rapidly position large numbers of FOVs using an intuitive, interactive graphical interface.

TSAI provides two methods for setting up FOVs. One method builds FOVs using only the optical image, while the other generates a tiled SED image upon which FOVs are built. The optical-only method operates within the same paradigm as the manufacturer's user interface - rough positioning using the optical image followed by precise positioning under the SED view. TSAI just accelerates the process with an interactive graphical layer (steps 5 and 6) and keyboard enhancements (step 7).

The tiled SED method (step 4) breaks the paradigm by providing precise positioning up front, obviating the need to finely adjust each FOV one-by-one using the SED view. Furthermore, users can perform light microscopy on an hematoxylin and eosin-stained serial section (adjacent slice of tissue), identify structures of interest, and more easily pinpoint their positions on the tiled SED image for MIBI imaging.

The critical factor in the tiled SED method is setting the correction coefficients to eliminate misalignments or, ideally, having the manufacturer calibrate the instrument such that all the coefficients can be set to 0. Large misalignments in tiled SED scans indicate that the coefficients need to be adjusted (Figure 3). Using patterned slide burns (steps 4.12.1 to 4.12.8, Supplementary Figure 6), one can quickly troubleshoot and perform coarse corrections before fine-tuning with subsequent tiled SED scans. This process for determining the coefficients typically takes several hours but only needs to be redone if the instrument hardware is modified. If the process is too troublesome to perform within an acceptable amount of time, one may default back to the optical-only method for setting up FOVs.

With regards to the code itself, TSAI has been tested on software versions 1.8 and 1.7 (specifically 1.8.2.1 and 1.7.0-0f60ffbc) and is immediately ready for use by hundreds of current MIBI users. As it stores settings in cookies, TSAI must reside on a website. Otherwise, it is written using HyperText Markup Language 5, JavaScript, and Cascading Style Sheets - existing standards already built into modern web browsers^13,14,15. Thus, it requires no installation of plug-ins, extensions, or external programs or compilers such as Matlab, Python, R, Juypter, or Docker. TSAI leverages very helpful features of the MIBI user control interface - a way to import/export run parameters from a JSON file (Figure 1C) and being built within a web browser. Since web browser coding is platform-independent, with extensive graphics source code libraries and populous communities of programmers, users can integrate their own code much more easily than they could for a traditional, proprietary, compiled, closed-source Windows or Unix interface. Thus, instrument manufacturers may wish to embrace browser-based interfaces more often, empowering users to extend them beyond what the manufacturer originally envisioned and thereby spur further innovation.

Materials

Name	Company	Catalog Number	Comments
MIBI computer	Ionpath
MIBIcontrol (software)	Ionpath
MIBIscope	Ionpath		Multiplexed Ion Beam Imaging (MIBI) microscope
MIBIslide	Ionpath	567001	Conductive slide for MIBI
Tile/SED/Array Interface (TSAI) (software)		https://github.com/ag-tsai/mibi_tsai/