Imaging cortical vasculature with stimulated Raman scattering and two-photon photothermal lensing microscopy

The ability to map microvascular morphology and hemodynamic parameters, such as blood volume, is desirable for many biomedical studies and will lead to a deeper understanding of the mechanisms of angiogenesis and vascular disease. Capillary networks can be delineated in three dimensions with two-photon excited fluorescence microscopy; however, this requires the intravenous infusion of a fluorescent dye into the blood plasma, which often complicates in vivo imaging and only provides an indirect estimate of local haematocrit volume. Moreover, visualising the spatial distribution of capillaries is often insufficient; ideally, one would wish to correlate the proximity of the blood vessels with the surrounding local tissue structure. We present in this study a novel multimodal approach that combines stimulated Raman scattering and two-photon photothermal lensing to provide simultaneous visualisation of cortical microvasular morphology and surrounding cellular structures. We show that volumetric analysis of the nonlinear photothermal contrast of erythrocytes allows a direct quantification of local haematocrit volume rather than relying upon average plasma volume-to-haematocrit ratios.