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 microarray is unwieldy using the manufacturer's interface. We developed the tiled SED array interface to help users rapidly position large numbers of FOVs using an intuitive interactive graphical interface.

To begin, open the TSAI web user interface in the web browser. Then open the Optical Coregistration menu and click Copy Automatic Coregistration Code to Clipboard. Open the MIBI user control interface in the web browser and press Ctrl+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. 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 tiles positioning, hover over each tile in the tile's 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.