An orange fluorescent glutamate sensor for multicolor single-synapse imaging

High-resolution imaging of glutamate is essential for understanding synaptic transmission. While fluorescent sensors capable of detecting glutamate release with single-synapse resolution have been developed, these sensors are almost exclusively green-emitting. This spectral constraint hinders multicolor imaging experiments designed to investigate the interplay between glutamate dynamics and other cellular activities. To overcome this spectral limitation, we engineered an orange-emitting fluorescent glutamate sensor, OR-EOS, by site-specific labeling of the glutamate binding domain of GluA2 with the synthetic dye Cy3. This sensor showed a robust fluorescence decrease upon glutamate binding, with a dissociation constant of approximately 3 μM. In cultured neurons, OR-EOS detected glutamate release evoked by a single action potential at individual synapses. The longer wavelength fluorescence of OR-EOS enabled dual-color imaging of glutamate release and presynaptic calcium influx at the same synaptic sites by combined use of a green calcium indicator. OR-EOS expands the spectral palette for functional synaptic imaging, providing a powerful new tool for dissecting complex neural mechanisms.