Youngseop Lee

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.

Microvascular function and oxygen metabolism are central to tissue and organ health. However, label-free methods for imaging oxygen dynamics in three-dimensional (3D) microvascular networks at the level of single red blood cells (RBCs)—the fundamental units of oxygen transport in vivo—remain lacking. Here, we introduce super-resolution functional photoacoustic microscopy (SR-fPAM), which spatiotemporally tracks RBC movements under dual-wavelength excitation. SR-fPAM reconstructs super-resolved 3D microvascular architecture comparable to two-photon microscopy while providing quantitative measurements of RBC flow and oxygenation. In live mice, SR-fPAM revealed redistribution of oxygen and hemodynamics across 3D microvascular networks following a single-vessel stroke. These findings establish SR-fPAM as an enabling tool that bridges a critical gap in oxygen-metabolism imaging and opens new avenues for studying microvascular health and disease with unprecedented functional insights.

Single-molecule localization microscopy (SMLM) enables super-resolution imaging on conventional fluorescent microscopes. Spectroscopic SMLM (sSMLM) further allows highly multiplexed super-resolution imaging. We report an easy-to-implement symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM design that maximizes photon utilization. We first symmetrically dispersed photons to the −1 ^st and +1 ^st orders in an optical assembly using two identical dual-wedge prisms (DWPs). Then we computationally extracted the fluorophores’ spatial position and spectral characteristics using photons in both the −1 ^st and +1 ^st orders. Theoretical analysis and experimental validation showed lateral and spectral precisions of 10.1 nm and 0.3 nm, respectively, representing improvements of 28% and 48% over our previous DWP-based system, where emitted photons are divided separately for spatial and spectral analyses.