# Multiplexed Nanoscale Protein Mapping Through Expansion Microscopy and Immuno-SABER

> **NIH NIH RF1** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $2,690,712

## Abstract

Tools for surveying brain cell types and circuits must be scalable, both in the number of molecular targets
visualizable at once, and in the size of the tissues that can be assessed. They also must be high resolution,
since cellular compartments such as axons, dendrites, and synapses exhibit nanoscale feature sizes. Despite
rapid progress by many teams in multiplexed imaging of expressed RNAs in intact brain circuits, or “spatial
transcriptomics”, technologies for multiplexed imaging of proteins intact brain circuits lag behind, despite the
fact that knowing the precise identity and location of proteins in defined synaptic, axonal, dendritic, and
subcellular compartments is one of the keys to understanding neural function, and thus deriving cell types for a
systematic census. Furthermore, given that many proteins are located in specific nanoscale compartments of
neurons, and many attain their full functionality only in the context of densely packed nanoscale complexes of
many proteins7, the need for nanoscale mapping is even more acutely felt for proteins than for RNAs. We here
propose to address these limitations by creating (Aim 1) a full toolbox for the multiplexed imaging of at least
30, and ideally 50, proteins at once, by optimizing the use of DNA-barcoded antibodies for rapid serial imaging
of many different neural proteins (aka Immuno-SABER, developed by the group of PI Peng Yin), in the context
of expansion microscopy, a radical new method for nanoimaging that utilizes physical expansion of the sample
(developed by the group of PI Ed Boyden). We will also develop, for the purposes of Immuno-SABER
multiplexed antibody imaging, a new form of expansion microscopy that decrowds proteins from one another,
for better access by antibodies (Aim 2). Finally, we will integrate the nanoscale, highly multiplexed, spatial
proteomics methods described above with spatial transcriptomics (Aim 3). We will integrate in situ sequencing
of expanded specimens, an existing project of the Boyden lab (manuscript in preparation), with the Immuno-
SABER protocol of Aim 1. In this way we will be able to simultaneously survey proteomic and transcriptomic
information, throughout neural architectures, with nanoscale precision. We will aim to deliver the ability to
survey at least 100 transcripts and 30 proteins, and ideally 150 transcripts and 50 proteins, in the same brain
specimen. We will validate and demonstrate the power of our technology in the context of our BICCN U19
collaborators' brain circuits of interest. Importantly, we are focusing from the beginning on the questions of
scale and accuracy, key to the success of the BICCN. We aim to deliver to the neuroscience community not
just a toolbox that is easy to use, but very powerful. Our toolbox will confront, head on, the limitations of
previous protein imaging strategies that lack scale in terms of numbers of proteins imageable, resolution, and
combinability with transcriptomics. Through regular meetings and...

## Key facts

- **NIH application ID:** 10088537
- **Project number:** 1RF1MH124606-01
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Edward S. Boyden
- **Activity code:** RF1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,690,712
- **Award type:** 1
- **Project period:** 2020-09-16 → 2024-09-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10088537

## Citation

> US National Institutes of Health, RePORTER application 10088537, Multiplexed Nanoscale Protein Mapping Through Expansion Microscopy and Immuno-SABER (1RF1MH124606-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10088537. Licensed CC0.

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