# Single-molecule and super-resolution imaging methods with maximum photon efficiency, increased spatiotemporal resolution and high detection sensitivity in densely crowded environments

> **NIH NIH R21** · SLOAN-KETTERING INST CAN RESEARCH · 2020 · $224,500

## Abstract

ABSTRACT
Reaching a more complete understanding of biological processes and mechanisms that
underlie health and disease demands a better integration of information spanning multiple
length and time scales. Super-resolution microscopy and single-molecule approaches have
emerged as potent tools that extend the spatial resolution and detection sensitivity in live
biological imaging. However, the current state-of-the-art techniques often achieve limited 3D
resolution that precludes visualizing spatial organization at the molecular scale. Moreover
balancing trade-offs between temporal and spatial resolution, while operating with a limited
photon budget often results in severely shortened single-molecule observation times. Finally,
many microscope configurations are challenged when imaging weak signals from single-
molecules, especially due to high background in crowded cellular specimens. Thus, although
promising, the full potential of single-molecule/super-resolution methods for transforming our
molecular understanding of biological processes has yet to be realized. To fill critical technical
gaps, new optimized microscope configurations are needed - that can operate at the limits of
spatiotemporal resolution while maximizing the information content of dim fluorescence signals.
We hypothesize that this goal can be achieved through novel combinations of 3D interferometry,
targeted fluorescence switching, while further harnessing emerging photon-efficient algorithms
to increase resolution as well as prolong total observation times. Based on these ideas we
propose to develop novel super-resolution and single-molecule fluorescence imaging tools,
focusing on two specific aims: (1) To extend the spatiotemporal scales of localization-based
single-molecule imaging and tracking to 1 nanometer isotropic 3D resolution and to ~1,000
data-point in vivo observation traces at down to (sub)millisecond sampling rates; (2) To achieve
real-time single-molecule detection sensitivity in addressable 3D volumes, at presence of micro-
Molar background concentrations, and inside highly crowded intracellular environments. The
new techniques will significantly increase our abilities to interrogate dynamic biological
processes with molecular detail, thus having widespread and immediate impact across
biomedical disciplines.

## Key facts

- **NIH application ID:** 10005376
- **Project number:** 5R21GM134342-02
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Alexandros Pertsinidis
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $224,500
- **Award type:** 5
- **Project period:** 2019-09-01 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10005376

## Citation

> US National Institutes of Health, RePORTER application 10005376, Single-molecule and super-resolution imaging methods with maximum photon efficiency, increased spatiotemporal resolution and high detection sensitivity in densely crowded environments (5R21GM134342-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10005376. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*