# Microflow time-resolved cytometry for FRET and fluorescent protein development

> **NIH NIH R01** · NEW MEXICO STATE UNIVERSITY LAS CRUCES · 2021 · $289,400

## Abstract

Project Summary
The goal of this research project is to design and apply fluorescence decay kinetic-based flow cytometry on a
microchip platform. The system will be used to quantify Förster resonance energy transfer (FRET) events
inside of mammalian cells and fully enrich near-infrared fluorescent proteins based on their photo-kinetics. The
microflow device will incorporate unique features such as acoustic focusing of cells through microfluidic
channels, multi-frequency measurements that give rise to multiple-fluorescence lifetime values per cell,
imaging capabilities to capture multi-pixel fluorescence lifetime measurements, and sorting capabilities
dependent on decay-kinetic parameters. Our first aim will be to use the cytometer to count cells based on
changes in the fluorescence (FRET) donor’s changing fluorescence lifetime. When FRET is evaluated by the
excited state kinetic changes of the energy-transferring fluorophore pairs, the result is a data set that has not
been affected by intensity-based artifacts. Moreover, with new computational toolboxes including phasor-based
FRET trajectories and FRET efficiency, cytometric parameters are developed for cell screening that provide
heterogeneity of lifetimes within the cell at a rate of thousands of cells per second. We test this with FRET at
the cell surface as well as with an intracellular FRET bioprobe. Both systems have biomedical significance
related to protein function alteration thereof with targets during screening. The second aim for this project is to
take the microchip-based system and use it to actively screen bacterial libraries and sort single cells that
express near-infrared fluorescent proteins with high quantum yield. The quantum yield is a photophysical trait
of fluorescent molecules that is directly proportional to the average fluorescence lifetime, or average time the
fluorophore spends in the excited state. Therefore a tool that can isolate samples based on the fluorescence
lifetime is quite valuable since the average intensity can be plagued by other factors such as concentration,
quantum efficiency, and instrument artifacts. The long term significance of our second aim is the ability to
expedite the development of near-infrared fluorescent proteins for use in molecular and diffuse optical
tomography. In general, the development of a compact, sensitive, and time-dependent cytometry system is
impacting beyond the two biomedical applications proposed. Accordingly this work is the first step toward
evaluating the benefits, demonstrating the quantitative nature, and setting the stage for broad use across many
more cytometric applications.

## Key facts

- **NIH application ID:** 10223368
- **Project number:** 5R01GM129859-04
- **Recipient organization:** NEW MEXICO STATE UNIVERSITY LAS CRUCES
- **Principal Investigator:** Jessica Perea Houston
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $289,400
- **Award type:** 5
- **Project period:** 2018-09-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10223368

## Citation

> US National Institutes of Health, RePORTER application 10223368, Microflow time-resolved cytometry for FRET and fluorescent protein development (5R01GM129859-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10223368. Licensed CC0.

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