# Engineering photostable fluorescent proteins and biosensors using transcriptomic mining and massive-throughput single-cell screening

> **NIH NIH R01** · BAYLOR COLLEGE OF MEDICINE · 2022 · $637,687

## Abstract

PROJECT SUMMARY/ABSTRACT
Fluorescent proteins are ubiquitous reagents in the biomedical sciences for reporting gene expression, protein and nucleic
acid localization, cell shape, and cellular activity. However, fluorescent proteins (FPs) become progressively dimmer —
they photobleach — with repeated or prolonged illumination. Photobleaching limits multiple types of biological experiments
where photostability is essential, such as single-molecule biophysics and timelapse imaging of cellular activity during
development, learning, and aging. Photobleaching often cannot simply be addressed by increasing the excitation light, as
high illumination power can induce membrane blebbing, nuclear fragmentation, alterations in the cell cycle, changes to the
concentration of intracellular calcium, and, ultimately, cell death. While over two decades of FP engineering has led to a
toolbox of bright FPs, less attention has been devoted to improving photostability because of the greater difficulty and lower
throughput endured when screening for photostable FPs. Moreover, few studies have attempted to improve photophysical
properties under two-photon illumination — a method of choice for deep-tissue imaging — because of technical challenges
associated with screening under this imaging modality. The overall objective of this research proposal is, therefore, to
develop and apply a color palette of bright and photostable FPs for one- and two-photon imaging in mammalian cells. Our
proposal leverages two specialized and synergistic approaches to FP discovery and engineering: (1) SPOTlight, a new all-
optical screening approach developed in Dr. St-Pierre's lab that circumvents technical hurdles and enables rapid screening
of both brightness and photostability at the single-cell level under one- and two-photon illumination; and (2) transcriptomic
and metagenomic mining for novel FPs from marine invertebrates, a technique pioneered by Dr. Shaner’s lab. SPOTlight
relies on light patterning technology to selectively illuminate individual cells labeled with fluorophores that can be
photoactivated from a dim to a bright state. The cells are therefore tagged with a unique fluorescence signature that can then
be distinguished and retrieved using Fluorescence Activated Cell Sorting (FACS). SPOTlight thus enables screening in
dense mixed cultures with single-cell resolution, thereby eclipsing the throughput of traditional well-based approaches.
Mining for novel FPs in marine invertebrate transcriptomes and metagenomes will allow us to rapidly identify and
characterize hundreds of novel FPs. From this pool of new FPs, we will select the most photostable for engineering with
the SPOTlight pipeline. We will also model their structures to guide site-directed mutagenesis. We propose to leverage
these new technologies and assays to develop FPs of different colors that are bright, monomeric, and sufficiently photostable
for long-term imaging experiments. We also propose to apply these ne...

## Key facts

- **NIH application ID:** 10422081
- **Project number:** 1R01EB032854-01
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Francois St-Pierre
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $637,687
- **Award type:** 1
- **Project period:** 2022-05-01 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10422081

## Citation

> US National Institutes of Health, RePORTER application 10422081, Engineering photostable fluorescent proteins and biosensors using transcriptomic mining and massive-throughput single-cell screening (1R01EB032854-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10422081. Licensed CC0.

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