Asynchronous-spectral fusion fluorescence microscopy for microsecond-scale behavioral dynamics.

Event-based image sensors provide microsecond temporal resolution but lack spectral discrimination, whereas diffractive spectral imagers encode wavelength information at conventional frame rates. We introduce a fluorescence microscopy architecture that fuses asynchronous event streams with diffraction-encoded CMOS measurements to decouple temporal and spectral sampling. The system achieves ~2.2 µm spatial resolution over a 0.5 mm field of view and effective temporal resolution down to 10 µs and differentiates fluorophores whose emission peaks are separated by only 23 nm. By synchronizing and computationally merging both sensing modalities, we enable spectrally resolved tracking capable of reaching 100,000 frames/s without scanning or filter switching. For the behavioral quantification of C. elegans presented in this work, we utilized a 100 µs integration window (10,000 frames/s) to maintain a high signal-to-noise ratio while resolving stimulus-evoked dynamics with sub-millisecond precision. This resolution is more than sufficient to capture the fastest recorded locomotion and response latencies, which occur on timescales of 10 to 100 milliseconds. Applied to live C. elegans, the platform resolves blue-light-evoked omega turns and reveals a significant genotype-dependent difference in response latency (0.18 s in wild type vs. 0.88 s in unc-101). These results establish asynchronous-spectral fusion as a general strategy for ultrafast, multiplexed fluorescence microscopy capable of quantifying rapid behavioral dynamics.