Adapting a Two-Photon Scanning Microscope for Simultaneous Single-Photon Imaging of an Infrared Dopamine Sensor.

We describe a novel method for adapting a two-photon scanning microscope to enable simultaneous detection of two-photon-generated visible fluorescence and single-photon-generated near-infrared (nIR) fluorescence. In this configuration, nIR fluorescence is routed through a single-mode optical fiber before detection by a photomultiplier tube. This fiber coupling offers two advantages: first, the optical fiber functions as a pinhole aperture, allowing for improved optical sectioning of the nIR signal; second, it minimizes nIR background fluorescence. To validate the effectiveness of this design, we conducted two sets of experiments in male and female C57B/6J mice. First, we compare two fluorescence indicators of the neurotransmitter dopamine: the genetically encoded indicator GRABDA and single-walled carbon nanotube-based optical nanosensors (nIRCats). Although nIRCats exhibit lower affinity for dopamine than GRABDA, this property allows for identification of high concentration release sites in the striatum. Second, we simultaneously imaged depolarization-induced calcium changes and dopamine release in the retina. Together, these results demonstrate the utility of integrating confocal nIR detection into a two-photon platform for simultaneous functional imaging across complementary spectral channels.