DUET: Motion-robust dual-color miniature two-photon microscope with low-crosstalk in freely behaving mice

We present dual-fiber excitation and temporal multiplexing (DUET), a dual-color miniaturized two-photon microscope (mTPM) integrating dual-fiber laser delivery and pixel-level temporal multiplexing. Two hollow-core photonic bandgap fibers with exceptional bend tolerance independently deliver 920 nm and 1,030 nm femtosecond pulses for fluorescence excitation, ensuring motion-robust imaging in freely moving mice. A custom 0.276°-wedged dichroic mirror corrects beam-pointing mismatch from dual-fiber delivery. Pixel-wise temporal gating eliminates excitation and emission crosstalk without sacrificing frame rate. During tail suspension tests, a paradigm inducing extreme motion, DUET achieves stable dual-color imaging of GCaMP6f- and tdTomato-expressing neurons at 8.4 Hz with negligible crosstalk. This enables continuous resolution of calcium dynamics from 123 excitatory and 10 inhibitory neurons throughout natural behavior. DUET establishes a motion-robust, high-fidelity dual-color mTPM for monitoring cell-type-specific neural interactions in vivo.