In recent studies, stimulated Raman scattering (SRS) and transient absorption microscopy (TAM) have been employed for label-free mapping of biomolecules (e.g., proteins and lipids) in brain tissues and hemoglobin in red blood cells, respectively. In this study, we combined SRS and TAM to simultaneously image cell densities and capillary structure in vivo at the highest reported imaging depth, ~300 µm, for either technique. This multimodal approach resulted in label-free identification of endothelial cells and pericytes in vivo with 90% accuracy using a machine learning classifier. Simultaneous two-photon excited fluorescence microscopy serving as the ground truth.
Hematocrit, the volume fraction of red blood cells in whole blood, is a crucial metric of animal health in pharmacokinetic and disease model experiments. While robust, current methods for measuring hematocrit can be prohibitory for longitudinal animal models where animal well-being restricts serial blood collection volumes. Here we present in vivo hemoglobin concentration quantification in a mouse ear model using bimodal ratiometric imaging of transient absorption of hemoglobin and stimulated Raman scattering of water. Additionally, we leverage the intrinsic high resolution of nonlinear imaging to demonstrate a low volume method for ex vivo size and volume quantification.
Blood oxygen saturation (sO2) 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 label-free 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.
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