Intravital microscopy (IVM) has been an invaluable tool for studying various mechanisms taken place in biological cells and tissues under natural physiological conditions including temperature, molecules gradient, oxygen, CO2 and pH concentration. IVM can be implemented by various imaging modalities including confocal scanning microscopy and multi-photon microscopy which measure excited light signals of fluorescent probes attached by target proteins. Since fluorescence imaging techniques measure light intensity, however, they have been lacking of quantitative information about biochemical and morphological properties of individual cells. In addition, since three-dimensional (3-D) images are obtained by axial stacking 2-D images, the limited acquisition speed hinders the video-rate 3-D imaging of cellular behaviors in live animals.
Here, we present a method to measure 2-D and 3-D quantitative phase imaging (QPI) of individual red blood cells (RBCs) flowing in the microvasculature of live mouse mesentery. QPI has been an emerging imaging technique which can measure optical phase delay of microscopic samples such as biological cells and tissues in label free. So far, various QPI techniques have been developed to study individual cells and tissues cultured in vitro, while in vivo QPI has remained unexplored due to multiple light scattering in tissues. We developed a post image-processing technique that remove the light scattering in tissues, and experimentally demonstrated that 2-D intravital QPI enables the investigation of the fluid dynamics of RBCs in capillaries of mouse mesentery and 3-D intravital optical diffraction tomography (ODT) measures chemical and morphological parameters of RBCs in capillaries. We expect the presented technique can be used for the in vivo investigation of various pathophysiological changes of RBCs inside micro-capillaries.