A cortex-wide multimodal microscope for simultaneous Ca²⁺ and hemodynamic imaging in awake mice

Developments in optical imaging techniques have advanced the study of neurovascular coupling across the whole cortex. Unfortunately, each cortex-wide optical imaging modality is specialized in revealing limited neural or hemodynamic actions. Here, we develop a cortex-wide multimodal microscope (multiScope) integrating widefield Ca²⁺ fluorescence microscopy, optical resolution photoacoustic microscopy, and laser speckle contrast imaging to simultaneously observe neuron firing, total hemoglobin and blood flow velocity with endogenous biomarkers. The multiScope features a cortex-wide field-of-view of Ø 8.6 mm, a maximum imaging speed of 4 Hz, and the average spatial resolutions of 10.7 ± 3.1 μm and 7.1 ± 0.8 μm respectively for widefield imaging and photoacoustic microscopy after model-based restoration. We demonstrate the multi-parametric imaging capability of the multiScope using animal models, observe fast neural and hemodynamic activities across the entire cortex, and carry out both global and local neural vascular coupling analyses for different brain stimulations.