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Updated: May 2, 2026

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Published on: October 8, 2014

An orange fluorescent glutamate sensor for multicolor single-synapse imaging.

Kenji Takikawa¹, Hirokazu Sakamoto², Daisuke Asanuma²

¹Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan; Division of Bioconvergence, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, 329-0498, Tochigi, Japan.

Biochemical and Biophysical Research Communications

|August 7, 2025

Summary

Researchers developed OR-EOS, an orange fluorescent sensor for glutamate, enabling multicolor imaging of synaptic transmission. This new tool allows simultaneous visualization of glutamate release and calcium activity at single synapses.

Keywords:

Glutamate sensor Hybrid-type sensor Multicolor imaging Orange fluorescence Single-synapse imaging

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Area of Science:

Neuroscience
Molecular Biology
Biochemistry

Background:

High-resolution imaging of glutamate is crucial for understanding synaptic transmission.
Existing fluorescent glutamate sensors are primarily green-emitting, limiting multicolor imaging capabilities.
Spectral constraints hinder investigations into the interplay between glutamate dynamics and other cellular processes.

Purpose of the Study:

To engineer a novel orange-emitting fluorescent glutamate sensor to overcome spectral limitations.
To enable multicolor imaging of synaptic activity by expanding the available spectral palette.
To provide a new tool for dissecting complex neural mechanisms.

Main Methods:

Engineered the orange-emitting fluorescent glutamate sensor OR-EOS.
Utilized site-specific labeling of the glutamate binding domain of GluA2 with the synthetic dye Cy3.
Validated sensor performance in cultured neurons, assessing glutamate binding affinity and detection of evoked release.

Main Results:

OR-EOS demonstrated a robust fluorescence decrease upon glutamate binding, with a dissociation constant of approximately 3 μM.
The sensor successfully detected glutamate release evoked by single action potentials at individual synapses.
Dual-color imaging of glutamate release and presynaptic calcium influx was achieved at the same synaptic sites.

Conclusions:

OR-EOS expands the spectral options for functional synaptic imaging.
The sensor facilitates simultaneous visualization of glutamate dynamics and other cellular activities.
OR-EOS serves as a powerful new tool for investigating complex neural mechanisms.