# Super-multiplexed fluorescence nanoscopy for imaging-based proteomics

> **NIH NIH R35** · UNIVERSITY OF CENTRAL FLORIDA · 2020 · $267,626

## Abstract

PROJECT SUMMARY
In situ immunofluorescence imaging is a powerful method to study the locations, expression levels and
structures of proteins in cells and tissues. In particular, multiplexed imaging reveals the interaction networks
of proteins, which allows us to understand the underlying mechanisms of many diseases. However, it has
been challenging to perform multiplexed immunofluorescence imaging due to its extremely time-consuming
process, high cost and lack of signal amplification. The limited spatial resolution achievable with confocal
microscopy often fails to reveal complex spatial organization and to determine localizations of proteins. Here
we propose super-multiplexed immunofluorescence nanoscopy that is capable of imaging more than twenty
different proteins in 24 hours with nanoscale resolution. We will employ DNA-barcoded secondary nanobodies
that are monovalent, open-source and designed for quantitative labeling. Repeated introduction and washing
of fluorescent DNA imagers will generate highly multiplexed images. Moreover, we will develop
unprecedentedly fast stimulated emission depletion (STED) microscopy that employs a parallelized line array
of doughnut beams. It will feature a large imaging area and excellent optical sectioning capability. Photon
reassignment, hyperspectral imaging and deep-learning will further facilitate rapid super-resolution-based
protein profiling. Our new biochemical and optical tools will play crucial roles in diverse biomedical areas
including brain proteomics and cancer profiling.

## Key facts

- **NIH application ID:** 10028050
- **Project number:** 1R35GM138039-01
- **Recipient organization:** UNIVERSITY OF CENTRAL FLORIDA
- **Principal Investigator:** Kyu Young Han
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $267,626
- **Award type:** 1
- **Project period:** 2020-09-15 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10028050

## Citation

> US National Institutes of Health, RePORTER application 10028050, Super-multiplexed fluorescence nanoscopy for imaging-based proteomics (1R35GM138039-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10028050. Licensed CC0.

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