# Extending the temporal and spatial capabilities of single-molecule methods

> **NIH NIH R01** · STANFORD UNIVERSITY · 2023 · $511,550

## Abstract

Project Summary / Abstract (30 line maximum)
 This research, in response to the PAR-19-253, “Focused Technology Research andDevelopment,”
aims to pioneer new advances in biological optical microscopy. Methods such as the development of
fluorescent proteins, single molecule fluorescence detection, single molecule fluorescence resonance
energy transfer (smFRET) and super-resolution microscopy enabled molecular level study of in vitro and
live cells of increasing complexity. The single molecule methods allowed researchers to observe kinetic
pathways and transient states unobservable with bulk methods. Despite recent advances, the existing
optical probes have limitations. Fluorescent proteins are comparable in size to the proteins they label and
photobleach quickly. In situ labeling of cytosol proteins is possible, but in vitro labeling methods are much
preferred and there are no reliable methods to introduce these proteins into cytosol of cells.
 This research will address these grand challenges by fundamentally expanding the toolbox of
optical microscopy. Aim 1 will develop new methods to introduce proteins labeled in vitro with organic
dyes directly into the cytosol of cells and the insertion of dye-labeled membrane proteins into cell
membranes, thereby expanding the application of optical probes to new biological systems. These
methods will be used to insert up-converting nanoparticle (UCNP) probes into live cells to allow the long-
term tracking of specific individual proteins from minutes to months with nanometer spatial resolution.
This technology will also allow the controllable transfection of cells with multiple genes. Aim 2 will
fundamentally improve the temporal resolution of smFRET to ≤ 100𝜇𝑠 and develop smFRET methods
that can span across cell membranes. Aim 3 will extend biological optical microscopy to access the
temporal and spatial scales of molecular motion. Here, UCNPs will be used to measure the continuous
transport of cargos by dynein in DRG neurons capable of resolving single molecular steps with one
millisecond time resolution over a distance of 900 𝜇𝑚. Using plasmonic optical probes, this work aims to
achieve ~ 100 𝑛𝑠 time resolution and < 1 𝑛𝑚 spatial resolution in live cells.
 By the end of the 4-year funding period, a device will be demonstrated that is able to introduce
controllable numbers of nanoparticles, proteins, and multiple genes and promoters into 1000s of cells with
high survival rates. The cells will be transferred onto microscope coverslips or microfluidic cells suitable
for high-resolution optical microscopy. An instrument capable of 100𝜇𝑠 smFRET will have been used to
study the dynamics of G-protein couped receptors (GPCRs). Another instrument will be built to improve
the time resolution of sub-nanometer movement to by up to ~ 100 𝑛𝑠. With this instrument, the real-time
visualization of the motion of molecular systems may be possible.

## Key facts

- **NIH application ID:** 10694885
- **Project number:** 5R01GM143554-03
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Steven Chu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $511,550
- **Award type:** 5
- **Project period:** 2021-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10694885

## Citation

> US National Institutes of Health, RePORTER application 10694885, Extending the temporal and spatial capabilities of single-molecule methods (5R01GM143554-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10694885. Licensed CC0.

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