H. Piyadasa was supported by the Canadian Institutes of Health Research (CIHR) Fellowship (MFE-176490). B. Oberlton was supported by the National Science Foundation (NSF) Fellowship (2020298220). A. Tsai was supported by a Damon Runyon Cancer Research Foundation (DRCRF) Fellowship (DRG-118-16), the Stanford Department of Pathology, the Annelies Gramberg Fund, and NIH 1U54HL165445-01. Additional acknowledgments go to Dr. Avery Lam, Dr. Davide Franchina, and Mako Goldston for helping to test and debug the program.

1. Loading of TSAI
<ol>
	<li>Run TSAI by opening&#160;https://tsai.stanford.edu/research/mibi_tsai in the web browser of the MIBI user control computer.
	<ol>
		<li>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&#160;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.</li>
	</ol>
	</li>
	<li>Alternatively, set up TSAI on any website with custom presets for any instrument.
	<ol>
		<li>Go to https://github.com/ag-tsai/mibi_tsai and download the mibi_tsai_standalone directory. Alternatively, download the&#160;Supplementary Coding File 1 .zip file and unzip the contents to a directory titled mibi_tsai_standalone.</li>
		<li>Open mibi_tsai_standalone/_resources/index.js in any text editor.</li>
		<li>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&#39;s settings. This mainly applies to customized instruments, but dwell time/timing choice pairs should be checked regardless. Save index.js.</li>
		<li>Upload mibi_tsai_standalone to any web server accessible through the internet, e.g., a lab web site or university-hosted web site.</li>
		<li>Open mibi_tsai_standalone/index.html in the web browser of the MIBI user control computer.</li>
	</ol>
	</li>
</ol>
2. Loading the MIBI slide and creating a template file
<ol>
	<li>Log into the MIBI experiment tracker (manufacturer-provided web interface for managing scan-related metadata) in the web browser.</li>
	<li>In the Slides tab, add&#160;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).</li>
	<li>In the Sections tab, add the new section to the panel (Supplementary Figure 2D).</li>
	<li>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).</li>
	<li>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.</li>
	<li>Export (download) the FOV list to a .json file (Supplementary Figure 3C). Download the optical image as a .png file (Supplementary Figure 3D).</li>
</ol>
3. Optical image-stage motor coregistration
<ol>
	<li>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.</li>
	<li>Open the Optical Coregistration menu. Click Copy Automatic Coregistration Code to Clipboard (Supplementary Figure 4A).</li>
	<li>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).</li>
	<li>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.</li>
</ol>
4. Tiled SED scan
<ol>
	<li>Load the optical image .png and .json files from step&#160;2.6&#160;by dragging and dropping them onto the TSAI web UI.</li>
	<li>Open the SED Tiler menu and click on a text box in the top row (Supplementary Figure 5A).</li>
	<li>Click (&#177;&#160;drag) on the optical image to select the top left corner for the SED scan (Supplementary Figure 5B).</li>
	<li>Press the D key or click on a text box in the second row in the SED Tiler menu.</li>
	<li>Click (&#177;&#8203;&#160;drag) on the optical image to select the bottom right corner for the SED scan.</li>
	<li>In the SED Tiler menu, click Copy SED Scan and Shift Correction Code to Clipboard (Supplementary Figure 5C).</li>
	<li>Open the MIBI user control interface in the web browser. Paste the code into the console and press Enter (Supplementary Figure 5D).</li>
	<li>Put the MIBI into SED mode on the QC - 300 &#181;m setting, move to an area that will not be acquired, and adjust the gain, focus, and stigmation.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Press Shift+T to start the tiled SED scan.</li>
	<li>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.</li>
	<li>If specific tiles are out of focus or otherwise improperly scanned, rescan them.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>To check SED-stage motor alignment, move to an area of the slide without tissue. Press Shift+5 to burn five 400 &#181;m FOVs in a checkerboard pattern (Supplementary Figure 6B-C) or Shift+9 to burn a 3 x 3 pattern of 400 &#181;m FOVs (Supplementary Figure 6D-E).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Press S to save a .png image of the pattern with the correction values in the file name.</li>
		<li>Drag and drop this .png file onto the TSAI web UI to load the values and save them to the browser cookie.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>If the tiled SED is adequate, press Escape. Return to the TSAI web UI.</li>
	<li>Drag and drop the tiled SED .png file onto the TSAI web UI (Supplementary Figure 5E).</li>
	<li>Click on the SED tab and adjust the zoom (Supplementary Figure 5F).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Tissue microarray (TMA)
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Click Build TMA from the TMA options menu (Figure 4K).</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>When move is selected, press 2, 4, or 8 to set the FOV size to 200 &#181;m, 400 &#181;m, or 800 &#181;m, respectively, and the raster dimensions will be scaled proportionately such that the imaging resolution remains unchanged.</li>
		<li>When move is selected, press A to go to the previous tile or&#160;press D to go to the next tile.</li>
		<li>To adjust other tile settings, click&#160;the&#160;&#8801; button to expand the settings menu if it is not visible.</li>
	</ol>
	</li>
</ol>
6. Area/polygon tile
<ol>
	<li>If setting FOVs to cover a contiguous area of tissue, first adjust FOV settings as necessary in the relevant tile of the Tiles&#160;column.</li>
	<li>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).</li>
	<li>Scroll to the bottom of the Tiles&#160;column and click the&#160;&#8801; button (^ when expanded, Figure 5E) in the new polygon tile to see the tile map.</li>
	<li>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.</li>
	<li>To toggle off multiple FOVs, click on Eraser and then click and drag on the tiled FOVs in the slide image (Figure 5H).</li>
	<li>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.</li>
	<li>To insert the rows above, click the &#9650; button (Figure 5I). To insert columns to the left, click the &#9668; button (Figure 5J).</li>
	<li>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.</li>
</ol>
7. FOV navigation and adjustment
<ol>
	<li>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.</li>
	<li>Open the FOV navigation/adjustment menu below the slide (optical or SED) image. Click Copy FOV Navigation Code to Clipboard.</li>
	<li>Open the MIBI user control interface in the web browser. Put the MIBI into SED mode and adjust the gain, focus, and stigmation.</li>
	<li>Press Ctrl+Shift+J to open the browser console, or right-click on the page and click Inspect, then open the Console tab.</li>
	<li>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.</li>
	<li>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.</li>
	<li>To adjust the SED magnification, press M (200 &#181;m), , (400 &#181;m), . (800 &#181;m), or / (maximum) keys.</li>
	<li>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.</li>
	<li>To toggle an FOV ON or OFF, press T. To change the FOV size, press 2 (200 &#181;m), 4 (400 &#181;m), or 8 (800 &#181;m). The raster dimensions will be scaled proportionately such that the imaging resolution remains the same.</li>
	<li>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.</li>
	<li>When satisfied, press D to go to the next FOV, or A to go back to the previous FOV. Repeat steps 7.6&#160;to 7.11&#160;for all FOVs.</li>
	<li>When finished with all FOVs, press X or Escape. Adjustments will be saved to a .txt file and copied to the clipboard.</li>
	<li>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.</li>
	<li>Click Adjust to apply adjustments to the tiles in the Tiles&#160;column.</li>
</ol>
8. JSON file generation and import
<ol>
	<li>Below the Tiles&#160;column, under Output, check the list of tiles and estimated run time (Supplementary Figure 7A).</li>
	<li>Under Group, select an option for FOV grouping (Supplementary Figure 7B). Grouping has no effect on the sequentially ordered .json file.
	<ol>
		<li>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.</li>
		<li>For the randomized .json file, do not group FOVs will randomly order FOVs such that FOVs from different tiles are intermixed.</li>
		<li>If in-run autofocus has been specified, FOVs will automatically be grouped by the closest autofocus site.</li>
	</ol>
	</li>
	<li>Under split, select an option for splitting into multiple .json files (Supplementary Figure 7C).
	<ol>
		<li>Do not split will keep all FOVs in only one .json file.</li>
		<li>Split by every # FOVs will split FOVs across multiple .json files, where each file contains the specified number of FOVs.</li>
		<li>Split by every # hours # minutes will split FOVs across multiple .json files, where each file&#39;s estimated run time is roughly the specified amount of time.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
</ol>

Accelerating MIBI Run Setup Using TSAI for Tissue Microarray Imaging

<ol>
	<li>Liu, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Ion+Beam+Imaging:+Insights+into+Pathobiology.">Multiplexed Ion Beam Imaging: Insights into Pathobiology.</a> Annu Rev Pathol. 17, 403-423 (2022).</li><li>Keren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIBI-TOF:+A+multiplexed+imaging+platform+relates+cellular+phenotypes+and+tissue+structure.">MIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure.</a> Sci Adv. 5 (10), 1-16 (2019).</li><li>Elhanani, O., Keren, L., Angelo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Dimensional+Tissue+Profiling+by+Multiplexed+Ion+Beam+Imaging.">High-Dimensional Tissue Profiling by Multiplexed Ion Beam Imaging.</a> Methods Mol Biol. 2386, 147-156 (2022).</li><li>Greenwald, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+segmentation+of+tissue+images+with+human-level+performance+using+large-scale+data+annotation+and+deep+learning.">Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning.</a> Nat Biotechnol. 40 (4), 555-565 (2022).</li><li>Risom, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+to+invasive+breast+cancer+is+associated+with+progressive+changes+in+the+structure+and+composition+of+tumor+stroma.">Transition to invasive breast cancer is associated with progressive changes in the structure and composition of tumor stroma.</a> Cell. 185 (2), 299.e18-310.e18 (2022).</li><li>McCaffrey, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunoregulatory+landscape+of+human+tuberculosis+granulomas.">The immunoregulatory landscape of human tuberculosis granulomas.</a> Nat. Immunol. 23 (2), 318-329 (2022).</li><li>Greenbaum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatially+resolved+timeline+of+the+human+maternal&#8211;fetal+interface.">A spatially resolved timeline of the human maternal&#8211;fetal interface.</a> Nature. 619 (7970), 595-605 (2023).</li><li>Hartmann, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+metabolic+profiling+of+human+cytotoxic+T+cells.">Single-cell metabolic profiling of human cytotoxic T cells.</a> Nat Biotechnol. 39 (2), 186-197 (2021).</li><li>Patwa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+imaging+analysis+of+the+tumor-immune+microenvironment+reveals+predictors+of+outcome+in+triple-negative+breast+cancer.">Multiplexed imaging analysis of the tumor-immune microenvironment reveals predictors of outcome in triple-negative breast cancer.</a> Commun Biol. 4 (1), 852 (2021).</li><li>Keren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Structured+Tumor-Immune+Microenvironment+in+Triple+Negative+Breast+Cancer+Revealed+by+Multiplexed+Ion+Beam+Imaging.">A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging.</a> Cell. 174 (6), 1373.e19-1387.e19 (2018).</li><li>Vijayaragavan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+spatial+proteomic+imaging+for+human+neuropathology.">Single-cell spatial proteomic imaging for human neuropathology.</a> Acta Neuropathol. Commun. 10 (1), 158 (2022).</li><li>. GitHub - angelolab/toffy: Scripts for interacting with and generating data from the commercial MIBIScope. (n.d.) Available from: <a target="_blank" href="https://github.com/angelolab/toffy">https://github.com/angelolab/toffy</a> (2023)</li><li>. HTML Living Standard Available from: <a target="_blank" href="https://html.spec.whatwg.org/multipage">https://html.spec.whatwg.org/multipage</a> (2023)</li><li>. ECMAScript 2022 Language Specification Available from: <a target="_blank" href="https://www.ecma-international.org/publications-and-standards/standards/ecma-262">https://www.ecma-international.org/publications-and-standards/standards/ecma-262</a> (2023)</li><li>. Cascading Style Sheets (CSS) Available from: <a target="_blank" href="https://www.w3.org/Style/CSS/Overview.en.html">https://www.w3.org/Style/CSS/Overview.en.html</a> (2023)</li></ol>

The authors declare no conflicts of interest.

Multiplexed ion beam imaging (MIBI) is a powerful technique for dissecting detailed cellular phenotypes and tissue histoarchitecture5,6,7,8,9,10,11. Computational efforts around MIBI have largely focused on processing the data after imaging, but little has been done to improve the instrument...

Multiplexed ion beam imaging (MIBI) is a technique to image 40+ proteins simultaneously on histologic tissue sections at up to 250 nm resolution1,2,3. 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 context1,2,3,4. 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 pregnancy5,6,7,8,9,10,11.
A key bottleneck in MIBI instrument operation is setting up fields of view (FOVs) - 200&#160;x 200 &#181;m2 to 800&#160;x 800 &#181;m2 areas of the tissue - for imaging. The MIBI images one FOV at a time, up to 800&#160;x 800 &#181;m2, 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 &#181;m (Figure 1B). First, the user roughly positions a single FOV using the optical image. Because the image resolution is only about 60 &#181;m per pixel, if the placement is off by two pixels (2 pixels&#160;x 60 &#181;m per pixel), a standard 400 &#181;m 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MIBI computer</td>
			<td>Ionpath</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIcontrol (software)</td>
			<td>Ionpath</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIscope</td>
			<td>Ionpath</td>
			<td></td>
			<td>Multiplexed Ion Beam Imaging (MIBI) microscope</td>
		</tr>
		<tr>
			<td>MIBIslide</td>
			<td>Ionpath</td>
			<td>567001</td>
			<td>Conductive slide for MIBI</td>
		</tr>
		<tr>
			<td>Tile/SED/Array Interface (TSAI) (software)</td>
			<td></td>
			<td>https://github.com/ag-tsai/mibi_tsai/</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid setup of tissue microarray and tiled area imaging on the multiplexed ion beam imaging microscope using the tile/sed/array interface 

Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique&#160;that generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in&#160;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.

Author Spotlight: Introducing the Tile/SED/Array Interface for Rapid Field of View Positioning in Tissue Imaging

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

Multiplexed ion beam imaging, or MIBI, is a technique to image protein expression in histological tissues across dozens of proteins simultaneously. Using MIBI, researchers can identify, locate, and characterize cells in their native tissue environments. A key bottleneck in MIBI instrument operation is setting up fields of view, or FOVs, for imaging.FOVs are 200 by 200 micron to 800 by 800 micron areas on the slide which users position to cover tissue regions of interest. Positioning FOVs for large contiguous areas of tissue and large tissue microarrays is unwieldy using the manufacturer's interface. We developed the tile/SED/array interface to help users this rapidly position large numbers of FOVs using an intuitive, interactive graphical interface.

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 coefficient...

Read this Scientific Article Protocol about Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope using the Tile/SED/Array Interface at JoVE.com

Multiplexed ion beam imaging (MIBI) is often used to image tissue microarrays and tiled, contiguous tissue areas, but current software for setting up these experiments is cumbersome. The tile/SED/array interface is an intuitive, interactive graphical tool developed to dramatically simplify and accelerate MIBI run setup.

Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique&#160;that generates 40+ plex images of protein ...

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Research

JoVE Journal

Biology

Optical Image-Stage Motor Coregistration and Tiled SED Scan Setup for Tile/SED/Array Interface (TSAI)

To begin, open the TSAI web user interface in the web browser. Then, open the optical coregistration menu and click copy automatic registration code to clipboard. Open the MIBI user control interface in the web browser and press Control Shift J to open the browser console.Paste the code into the console and press enter. Click the link generated in the console to load the coregistration into the TSAI web user interface and save it as a cookie. Drag and drop the optical image PNG and JSON files onto the TSAI web user interface.Open the SED tiler menu and click on a text box in the top row. Click and drag on the optical image to select the top left corner for the SED scan. Press the D key, then click and drag on the optical image to select the bottom right corner for the SED scan.In the SED tiler menu, click copy SED scan and shift correction code to clipboard. Next, open the MIBI user control interface in the web browser. Paste the code into the console and press enter.Put the MIBI into SED mode on the QC 300 setting, and move to an area that will not be acquired. Then, adjust the gain, focus, and stigmation. Press Shift T to start the tiled SED scan.Upon completion, the system automatically saves a new PNG file of the tiled SED image. Next, inspect the tiled SED scan for large misalignments. When tiled SED is adequate, press escape and return to the TSAI web user interface.Drag and drop the tiled SED PNG file onto the TSAI web user interface. Click on the SED tab and adjust the zoom. Use the slide options menu above the SED image to adjust line thickness, and cursor size, and brightness, and contrast.A properly tiled SED image shows minimal misalignments between SED tiles. In contrast, the suboptimal setup results in large misalignments between SED tiles.

Optimizing Tissue Microarrays and Area/Polygon Tiles for Efficient Large-Scale Imaging

To set a field of views or FOVs for a grid of tissue microarray or TMA spots, open a tiled SED image. In the relevant tile of the tiles column, adjust the columns and rows. Then check or uncheck the boxes in the map.In the relevant tile, click TMA to open the TMA Options menu. Set the number of rows and columns of TMA spots. If necessary, add a naming prefix and edit the starting row and column numbering.On the slide image, click on the four corners of the TMA. Then click and drag the circled corners to adjust the positioning of the crosshairs to best match the TMA spots. From the TMA Options menu, click Build TMA.To check the tile's positioning, hover over each tile in the Tiles column. Click Move to adjust the tile's positioning. Then click and drag on the slide image.To set FOVs to cover a contiguous area of tissue, first adjust FOV settings in the relevant tile of the tile's column. In the relevant tile, click Polygon to cover a contiguous area of tissue. Then click on the slide image to set the vertices and corners of the area to be tiled.Double-click to close the polygon and cover the area with FOVs. Scroll to the bottom of the Tile column and click the three stacked lines button in the new polygon tile to see the tile map. Click on Eraser to toggle off multiple FOVs.Then click and drag on the tiled FOVs in the slide image. To toggle on multiple FOVs, click on Clicker. Then click and drag on the empty areas in the slide image covered by the tile map.To insert the rows above, click the up arrow button. Click the left arrow button to insert columns to the left. To adjust tile positioning, click Move.Then click and drag on the slide image.

304 Views.  Stanford University.  Multiplexed ion beam imaging (MIBI) is often used to image tissue microarrays and tiled, contiguous tissue areas, but current software for setting up these experiments is cumbersome. The tile/SED/array interface is an intuitive, interactive graphical tool developed to dramatically simplify and accelerate MIBI run setup.

Accelerating MIBI Run Setup Using TSAI for Tissue Microarray Imaging

Summary