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

Summary

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.

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 Run TSAI by opening https://tsai.stanford.edu/research/mibi_tsai in the web browser of the MIBI user control computer. 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. Alternatively, set up TSAI…

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 coefficient…

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…

Materials

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/