In Vitro Quantification of Single Red Blood Cell Oxygen Saturation by Femtosecond Transient Absorption Microscopy

Hemoglobin, the oxygen carrying protein, ferries nearly all bodily oxygen from the lungs to cells and tissues in need. Blood oxygen saturation (sO₂) thus plays an important role in maintaining energy homeostasis throughout the body. Clinical and research tools have been developed to monitor sO₂ at a wide range of temporal and spatial scales. However, real-time quantification of sO₂ at single red blood cell (RBC) resolution remains challenging. Such capability is critically important to study energy metabolism in heterogeneous tissues including brain and tumor tissue. In this work, we develop a ratiometric transient absorption microscopy technique to image hemoglobin sO₂. By exploiting differences in transient lifetime kinetics between  oxyhemoglobin and deoxyhemoglobin, we directly quantified the sO₂ of single RBCs in real-time without the need for injection of exogenous agents. This simple and high-speed nonlinear optical imaging technique is well suited for in vitro and in vivo quantification of sO₂.