# Developing a strategy for 4-color in vivo two-photon imaging

> **NIH NIH R21** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2022 · $217,169

## Abstract

Many neurodevelopmental and neurodegenerative brain disorders manifest impaired synaptic integrity, stability,
and experience-dependent selection, resulting in wiring deficits and perturbed function. Yet our ability to
investigate how such disorders affect synaptic structure and function is severely limited by the difficulty of
visualizing synapses in the living brain and tracking their varied protein components. We propose developing
and testing new labeling and microscope configurations that would enable simultaneous live tracking of up to
four cellular proteins in the context of the intact mouse brain. The aims below put forth two 4-color constellations
that are designed to address distinct classes of experimental questions. Adjustments to the two-photon
microscope design that could accommodate both aims would primarily be in the detection path. Aim 1: To
develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to
four proteins situated on both sides of the synaptic cleft. The 4-color constellation in this aim is designed to
enable in vivo monitoring of a presynaptic afferent label in addition to two postsynaptic proteins and a
postsynaptic cell fill. We will label two excitatory post-synaptic markers that are considered mutually exclusive,
PSD95 and PSD93, in the context of a thalamic afferent genetic label. In this scenario, tdTomato labels thalamic
afferents, eYFP serves as a cell fill, PSD95-teal labels mature excitatory synapses, and PSD93 fused to a far
red fluorophore (iRFP682) labels immature excitatory synapses. With this labeling one could ask questions such
as, what is the ratio and dynamics of thalamic innervation to mature PSD95 positive spines vs immature PSD93
positive spines? This 4-color combination could also be used to monitor any two postsynaptic labels in
combination with any cell-type specific afferent label. Aim 2: To develop and implement spectrally resolved
two-photon microscopy for simultaneous tracking of up to four postsynaptic fluorophores, one of these
being green. There are several categories of fluorophores in the green range that would be particularly useful
to combine with structural markers. Most notably, the pH-sensitive GFP mutant, Super ecliptic pHluorin (SEP)
used to tag and track synaptic receptors, and the GCaMP family of calcium sensors, broadly used to monitor
neuronal activity. Unfortunately, a green fluorophore is incompatible with most blue and yellow labels due to
overlap of their emission spectra. Here pyramidal neurons would co-express: a red cell fill (mScarlet) to label
dendritic morphology, PSD95-iRFP682 and PSD93-BFP (a short wavelength blue) to label mature and immature
excitatory synapses, respectively, and SEP-GluR1. We could then ask questions such as, what are the dynamics
of AMPA receptor insertion into mature PSD95 positive vs immature PSD93 positive spines? This 4-color
combination could also be used to tag and track NMDA or GABA receptor subuni...

## Key facts

- **NIH application ID:** 10459675
- **Project number:** 1R21NS125396-01A1
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Elly Nedivi
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $217,169
- **Award type:** 1
- **Project period:** 2022-03-01 → 2024-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10459675

## Citation

> US National Institutes of Health, RePORTER application 10459675, Developing a strategy for 4-color in vivo two-photon imaging (1R21NS125396-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10459675. Licensed CC0.

---

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