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. First, we compare two fluorescence indicators of the neurotransmitter dopamine: the genetically encoded indicator GRAB_DA and single walled carbon nanotube based optical nanosensors (nIRCats). Although nIRCats exhibit lower affinity for dopamine than GRAB_DA, 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.