# Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy

> **NIH NIH R43** · PANLUMINATE INC. · 2022 · $248,545

## Abstract

Abstract
The brain is the most complex organ in the human body. Understanding its neural connections and molecular
anatomy requires imaging technology that is capable of mapping the 3D nanoscale distribution of specific
proteins in the context of brain ultrastructure. The currently best available option is correlative light and
electron microscopy (CLEM), which combines the resolving power and global contrast of EM with the high
molecular specificity of fluorescence microscopy. While very powerful, CLEM is laborious, low throughput,
expensive and often inadequate in spatial correlation resolution. To date, there is no imaging technology
that can resolve specific proteins within the ultrastructure of a synapse. We recently developed an
all-optical method called pan-Expansion Microscopy (pan-ExM) that has the potential to allow standard
confocal microscopy users to do just that: by combining ~20-fold linear expansion of biological samples with
novel bulk (pan) staining of total proteins, hallmark ultrastructural features such as post- and pre-synaptic
densities and mitochondria cristae can now be resolved by their characteristic pan-staining pattern, analogous
to heavy-metal stains in EM. Panluminate Inc. is a new company built around this core technology. Our
proposed Phase I project is to validate pan-ExM protocols that will establish pan-ExM as a brain imaging
technology. Our overall goal is to enable every neuroscientist to locate specific protein labels within their 3D
contextual compartments as well as perform EM-like neuronal tracing in brain tissue. We specifically propose
to (1) validate and optimize antibody-labeling compatible protocols for synapse identification, and (2) optimize
and validate a novel lipid pan-stain we developed to delineate cellular boundaries to complement our proteome
pan-stain. Ultimately, the developments proposed here will enable Panluminate to streamline and deploy
pan-ExM sample preparation kits to every neuroscientist interested in simultaneous 3D ultrastructural analysis,
antibody labeling, and neuronal tracing in brain tissue and to offer pan-ExM sample preparation and imaging as
a service.

## Key facts

- **NIH application ID:** 10483978
- **Project number:** 1R43MH128999-01A1
- **Recipient organization:** PANLUMINATE INC.
- **Principal Investigator:** Ons M'Saad
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $248,545
- **Award type:** 1
- **Project period:** 2022-09-16 → 2023-09-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10483978

## Citation

> US National Institutes of Health, RePORTER application 10483978, Development of a sample preparation protocol for brain ultrastructural analysis, immunolabeling, and neuronal tracing by light microscopy (1R43MH128999-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10483978. Licensed CC0.

---

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