Integrated two-photon and photoacoustic microscopy for single-cell neurometabolic imaging

Understanding how neuronal activity couples with local energy metabolism is fundamental to brain function. Oxygen exchange between individual neurons and red blood cells (RBCs) is central to this process, yet no existing method can simultaneously capture their dynamics at single-cell resolution in vivo. Here, we introduce integrated two-photon and photoacoustic microscopy (TPM-PAM), which enables real-time imaging of single-neuron calcium activity alongside oxygen release from individual RBCs in awake mice. In TPM-PAM, a transparent micro-ring resonator-based ultrasound sensor breaks the long-standing tradeoff between optical access and acoustic sensitivity, while dual-wavelength kymography simultaneously quantifies single-RBC oxygenation and flow to derive the oxygen release rate. Incorporating nonlinear optical manipulation of the neurovascular unit with cellular precision, TPM-PAM reveals distinct neurometabolic responses to whisker stimulation, single-capillary occlusion, and single-neuron stimulation. This work establishes a powerful platform for dissecting neurometabolic coupling at the cellular scale and understanding oxygen-metabolic regulation in brain health and disease.