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/6 mice. 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. Significance Statement Dual-color, real-time imaging is a powerful technique in biomedical imaging, including neuroscience. Here, we present a widely applicable modification to a standard two-photon scanning microscope that adds a near-infrared detection capability, a wavelength range that minimizes photon scattering and autofluorescence from biological samples. Using this microscope, we demonstrate the first direct comparison of two dopamine sensors: the genetically encoded sensor GRAB DA3m detected in the visible channel and carbon-nanotube–based sensors detected in the near-infrared channel. We further demonstrate simultaneous imaging calcium activity and dopamine signaling in the developing retina. While we focused on dopamine sensors in this study, this platform is broadly applicable to a wide range of fluorophores and can be implemented on existing two-photon microscopes.
Tarchick et al. (2026) studied this question.