# Ultra-high resolution, multiplexed single molecule nanoscopy and functional characterization of neural circuits

> **NIH NIH RF1** · PURDUE UNIVERSITY · 2020 · $2,424,695

## Abstract

The complex behaviors of all vertebrates are determined by the brain where neurons are connected by
synapses. The average volume of synapses corresponds to a sphere of ~400 nm radius—a size scale that can
barely be resolved using conventional optical microscopy methods. Synapses are tightly packed with molecular
assemblies of synaptic vesicles, synaptic and cytoskeletal proteins and neurotransmitter receptors. These
protein complexes are assembled in a much smaller scale—15-60 nm in the three-dimensional tissue space.
Current state-of-the-art imaging methods including optical super-resolution microscopy and expansion
microscopy remain limited in terms of its achievable resolution in 3D, resolution deterioration in thick tissues,
multiplexing capability and the access to the link between functional and structural connectivities. We propose
to develop a light-sheet illuminated, adaptive optics assisted, ultra-high resolution (10-15 nm 3D resolution)
4Pi/interferometric single-molecule super-resolution nanoscopy system for thick tissue specimens (Aim 1). We
will perform in vitro functional optogenetic neural circuit mapping using automated patch clamp followed by the
imaging of the same brain slices using our novel nanoscopy system to autonomously trace and resolve probed
circuit (Aim 2). To provide the highly multiplexed in situ protein detection capability (up to 8 targets), we will
combine Exchange-PAINT that utilizes DNA-barcode conjugated antibodies with the developed system (Aim
3). Our system will allow comprehensive mapping of ultrastructural features of neuronal circuits (such as pre-
and postsynaptic proteins in different types of synapses) in an functionally analyzed brain tissue with 10-15 nm
optical resolution in all three dimensions throughout a cortical microcircuit.

## Key facts

- **NIH application ID:** 10025878
- **Project number:** 1RF1MH123401-01
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Alexander Chubykin
- **Activity code:** RF1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,424,695
- **Award type:** 1
- **Project period:** 2020-08-20 → 2024-08-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10025878

## Citation

> US National Institutes of Health, RePORTER application 10025878, Ultra-high resolution, multiplexed single molecule nanoscopy and functional characterization of neural circuits (1RF1MH123401-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10025878. Licensed CC0.

---

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