# Development of a Versatile Multiplexing Nanoscopy Platform for Cell Biology

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $621,445

## Abstract

Project Summary
Understanding cellular function is intimately linked with the ability to visualize organelle ultrastructure with
molecular specificity and to observe how it is altered in diseases such as cancer, neurological disease,
ciliopathies and microbial pathogenesis. Super-resolution microscopy (SRM) has potential here as it bridges
the gap between light and electron microscopy and provides molecular specificity. However, SRM mostly offers
only a few color channels. This prohibits a comprehensive architectural map of organelles, as many are
pleomorphic and exist in multiple states depending on intra- and extracellular cues, making the combination of
datasets, each showing different subsets of labels, difficult. The SRM technique of DNA-PAINT allows, in
principle, powerful multiplexing to image 10 or more labels in one sample, but hurdles in speed, cost and ease
of use have limited its application. What is needed is a highly versatile multiplexing strategy to enable SRM of
organelles with an order-of-magnitude improvement in four key areas: acquisition speed, switching between
multiplex probe sets, spatial resolution, and cost. This requires new probes, instrumentation, enhanced
analysis, and biological validation. We will approach these tasks through three Specific Aims: 1) the
development of new versatile, DNA-PAINT probes that are both fluorogenic and provide a fast, adaptable,
low-cost framework for multiplexing, 2) a new platform for automated acquisition of multiplex DNA-PAINT data
and analytics to ‘connect the dots’ of single-molecule localization points in three dimensions and thereby create
membrane representations of organelles, and 3) the development of multiplexed DNA-PAINT ‘organelle
modules’ to validate this technology under realistic biological conditions and lower the entrance hurdle for
future biological users. Achieving these aims and their concrete deliverables will have a wide impact on the use
and accessibility of SRM to accelerate biological discovery.

## Key facts

- **NIH application ID:** 10753760
- **Project number:** 1R01GM151829-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Joerg Bewersdorf
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $621,445
- **Award type:** 1
- **Project period:** 2023-09-25 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10753760

## Citation

> US National Institutes of Health, RePORTER application 10753760, Development of a Versatile Multiplexing Nanoscopy Platform for Cell Biology (1R01GM151829-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10753760. Licensed CC0.

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