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 boundarie....

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 d....

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

List of materials used in this article

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/