# Bioorthogonal probe development for highly parallel in vivo imaging

> **NIH NIH R33** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $395,039

## Abstract

ABSTRACT
Multiplexed analytical techniques such as flow cytometry, scRNAseq and spatial transcriptomics have
revolutionized modern biology. Unfortunately, there are still no reliable ways of performing highly multiplexed in
vivo real-time imaging to understand the functional behavior of cells and molecules in their intact
microenvironment over time. Based on advances in “click chemistries” we have developed a novel, scission
based in vivo multiplexing technology (SAFE; Ko et al., 2022, Nat Biotech, in press; Ko et al., Adv Science,
2022 in press), that for the first time allows intravital imaging far beyond what is currently possible (~12-20
channel in vivo imaging in feasibility studies instead of 4-6 channels afforded by intravital microscopy). The
new technology utilizes specially labeled fluorescent affinity ligands i) where an exogenously administered
tetrazine cleaves a C2TCO containing linker between antibody and fluorochromes in seconds and at non-toxic
concentrations and ii) allows antibody-tagged superbright polymeric and indocyanine dyes to be washed away
between cycles of imaging and thus re-staining. The goal of this proposal is to further develop and validate the
technology and then use it to gain deeper temporal and spatial insight into the tumor microenvironment. We
propose two specific aims: i) Optimize the SAFE-IVM technology for broader (~30 channel) multiplexed in vivo
imaging (new chemistry including novel iTCO, Tz-BHQ, expand colors, improve imaging chambers); and ii)
perform multiplexed imaging of tumor microenvironment and its immune cells. Findings from latter studies will
be important because they will i) establish the utility of the new technology benchmarked against gold
standards and ii) open the door to broader applicability of the new technology.

## Key facts

- **NIH application ID:** 10831463
- **Project number:** 5R33CA277820-02
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** RALPH WEISSLEDER, MD, PhD
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $395,039
- **Award type:** 5
- **Project period:** 2023-04-21 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10831463

## Citation

> US National Institutes of Health, RePORTER application 10831463, Bioorthogonal probe development for highly parallel in vivo imaging (5R33CA277820-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10831463. Licensed CC0.

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