# Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells

> **NIH NIH R01** · UNIVERSITY OF MIAMI CORAL GABLES · 2023 · $90,524

## Abstract

PROJECT TITLE
Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells
PROJECT ABSTRACT/SUMMARY
The goal of our project is to develop synthetic dyes with photoactivatable fluorescence for the simultaneous
tracking of multiple structurally-distinct intracellular components in live cells. Specifically, the proposed studies
will lead to the realization of a palette of photoactivatable fluorophores (PAFs) that can be photoactivated with
mild green illumination (>500 nm) to produce partially-resolved fluorescence across the red region (>600 nm) of
the electromagnetic spectrum. Their photoactivation conditions will ensure negligible photodamage to live cells,
which instead cannot be avoided under the harsh irradiation required to operate existing PAFs. The high
brightness, infinite contrast and high photobleaching resistance engineered into our PAFs will enable the
localization of individual photoactivated molecules with precision at the nanometer level (≤20 nm) and their
tracking with millisecond response (≤10 ms) for several seconds (≥1 s) on the basis of single-particle tracking
photoactivated localization microscopy (spt-PALM). Their spectrally-resolved fluorescence will permit the
identification of structurally-distinct probes with the acquisition of emission spectra at the single-molecule level,
relying on spectroscopic single-molecule localization microscopy (sSMLM). Such a unique combination of
photochemical and photophysical properties is unprecedented and, in conjunction with established strategies to
label selectively different intracellular components of live cells with synthetic dyes, will allow the simultaneous
monitoring of multiple structurally-distinct targets with the characteristic high-throughput of spt-PALM and
spectral discrimination of sSMLM. The spatial resolution possible with our technology cannot be achieved with
conventional fluorescence imaging protocols and its high-throughput multiplexing capabilities cannot be
implemented in live cells with the many synthetic dyes and fluorescent proteins developed so far. Thus, the
innovative synthetic constructs that will emerge from the proposed studies can contribute to the investigation of
the fundamental factors governing cellular processes with multiplexing and super-resolution capabilities that are
not accessible with current fluorescent probes and imaging schemes.

## Key facts

- **NIH application ID:** 10794007
- **Project number:** 3R01GM143397-02S1
- **Recipient organization:** UNIVERSITY OF MIAMI CORAL GABLES
- **Principal Investigator:** Francisco M Raymo
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $90,524
- **Award type:** 3
- **Project period:** 2022-05-01 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10794007

## Citation

> US National Institutes of Health, RePORTER application 10794007, Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells (3R01GM143397-02S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10794007. Licensed CC0.

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