# New Probe and Methods for Correlated LM & EM

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2020 · $566,612

## Abstract

PROJECT SUMMARY
We propose to refine and exploit powerful new markers and labeling systems to visualize multiple proteins or
other biomolecules by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most
powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally
applicable genetically encoded labels until our recent development of new EM-compatible markers such as
miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM, and APEX, an
engineered ascorbate peroxidase. We have developed split-miniSOG, two fragments that do nothing
separately, but when brought back together reversibly regenerate miniSOG and its fluorescence and
photooxidative capability. Our newly developed split-miniSOG complementation system allows us to visualize
intermolecular interactions at high resolution by EM.
 We have developed new strategies and techniques to improve the acquisition of element-specific analytical
maps in transmission EM to achieve what we refer to as multicolor EM. This technology allows distinct EM-
level labeling of multiple species with a different lanthanide element that is separately imaged by electron
energy-loss spectrometry (EELS) and displayed in a distinct pseudocolor. Just as multicolor fluorescence has
been vital to understanding many cellular functions at optical resolution, we anticipate multicolor EM will be
valuable at finer resolution.
 Our overall goals are to expand and improve EM-compatible reporters, `molecular painting' chemistry, and
new instrumentation to improve the resolution, sensitivity, and specificity with which multiple proteins or other
biomolecules can be imaged by EM. We aim to obtain a genetically encoded far-red or near-infrared
diaminobenzidine (DAB) photooxidizer analogous to miniSOG but excited at significantly longer wavelengths to
facilitate multispecies labeling at high resolution. We will develop controlled living polymerization as an
alternative to photooxidative amplification using modern methods such as ATRP, ROMP, or RAFT applied to
fixed cells and tissue, to generate lanthanide-containing polymers of defined length and morphology at desired
cellular targets. As test cases, these techniques will be applied to fundamental biological problems such as the
spatial organization of the genome in the nucleus and aggregation of specified proteins involved in
neurodegenerative diseases. We have chosen these biological processes because they are diverse, engage
outstanding collaborators, and have great biomedical importance.
 Ultimately, the combination of photooxidizing, peroxidase-based, and nonphotochemical amplifying
systems will give cell and molecular biologists a rich palette for EM, comparable to small-molecule
fluorophores plus fluorescent proteins for optical microscopy.

## Key facts

- **NIH application ID:** 9923666
- **Project number:** 5R01GM086197-12
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Stephen Roy Adams
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $566,612
- **Award type:** 5
- **Project period:** 2008-09-30 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9923666

## Citation

> US National Institutes of Health, RePORTER application 9923666, New Probe and Methods for Correlated LM & EM (5R01GM086197-12). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/9923666. Licensed CC0.

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