# Methods for serially multiplexed labeling in EM reconstructions of brain tissue

> **NIH NIH R21** · UNIVERSITY OF CONNECTICUT STORRS · 2020 · $241,500

## Abstract

Project Summary/Abstract
 Tremendous technological progress in the use of serial electron microscopy (EM) for brain circuit
mapping has been made over the last decade, and it is possible to directly visualize synaptic connectivity in
larger volumes of brain tissue than ever before. Meanwhile, advances in molecular biology have shed new
light on the diversity among neurons, particularly with respect to their patterns of gene expression. Currently,
there are no methods available to efficiently integrate molecular labels into serial EM reconstructions. The
ability to distinguish many molecular cell type markers in serial EM volumes would greatly enhance our ability
to study circuit function, neuronal diversity, and neuroplasticity, and to determine how these are affected in
disease states. The major barrier to visualizing molecules in serial EM is that the methods used to preserve
morphology and generate contrast for serial EM are incompatible with most labels. Several methods have
been developed to accommodate this limitation, mainly by using genetic tools to introduce labels before tissue
samples are prepared for EM. These approaches are restrictive in that only a few labels can be used in a
single sample, genetic manipulation is required, and endogenous molecules cannot be localized. This project
will develop approaches that allow many different molecular labels to be differentiated within a single serial EM
tissue volume. An innovative strategy will be used: instead of working around the standard serial EM protocol
by designing labels that are compatible with it, the focus will be on replacing the incompatible elements of the
standard protocol. Circuit reconstruction by serial EM requires a high degree of morphological preservation,
which is typically accomplished with harsh chemicals that damage and denature molecules. However, all that
is fundamentally required to preserve morphology is to retain as many molecules in the tissue as possible,
which is also necessary for molecular labeling. Therefore, tissue preservation protocols will be developed to
minimize extraction of molecules without damage or irreversible denaturation. A combination of strategies will
be employed, including novel combinations of chemical crosslinkers and embedding resins. These new
approaches will offer a means of revealing valuable information about circuit organization and neuronal
diversity that is presently inaccessible.

## Key facts

- **NIH application ID:** 9892040
- **Project number:** 5R21MH119517-02
- **Recipient organization:** UNIVERSITY OF CONNECTICUT STORRS
- **Principal Investigator:** LINNAEA E OSTROFF
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $241,500
- **Award type:** 5
- **Project period:** 2019-03-13 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9892040

## Citation

> US National Institutes of Health, RePORTER application 9892040, Methods for serially multiplexed labeling in EM reconstructions of brain tissue (5R21MH119517-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9892040. Licensed CC0.

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