Label free high resolution in vivo optical imaging of microvessels

We have demonstrated a new optical microscopy technique for imaging microvasculature without any labeling. With a very sensitive two-color excited state absorption (ESA) measurement method, we demonstrated that oxy-hemoglobin and deoxy-hemoglobin show distinct excited state dynamics. Since this is a collinear measurement, we can readily apply it to the microscopic study of biological tissue. We have already demonstrated in vivo imaging of blood vessels in the nude mouse ear. Here we optimized the excitation and detection pulse train toward longer wavelengths, where tissue scatters less and greater penetration depth can be obtained. More importantly, we are able to separate arterioles from venules by employing different pump and probe wavelength combinations. This provides a powerful method to image blood vessels and their oxygenation level at the same time with micrometer resolution.