Localization optoacoustic tomography (LOT) in vivo with particles smaller than red blood cells

Abnormal microcirculatory changes in arterioles, venules and capillaries are key functional indicators of important diseases such as cancer, diabetes or cardiovascular disorders, as well as several neurodegenerative ailments. In vivo visualization of these structures has traditionally been restricted to the shallow depths <1-2 mm reachable with highresolution optical imaging methods. Optoacoustic (photoacoustic) tomography has enabled breaking through this light diffusion barrier to map functional hemodynamic parameters such as oxygen saturation in deep-seated vessels, but frequency-dependent attenuation of ultrasound waves limits the achievable resolution and prevents reaching the capillary level. Imaging of individual capillaries has been achieved via localization of microbubbles flowing in blood with pulseecho ultrasound. Optoacoustic localization of light-absorbing particles in mammalian tissues is however hampered by the strong background absorption of blood. Herein, we demonstrate the feasibility of in vivo localization optoacoustic tomography (LOT) with intravenously injected 5microdroplets having optical absorption 10000 higher than red blood cells for near-infrared wavelengths. Three-dimensional imaging of the mouse brain microvasculature through the intact scalp and skull was facilitated by the unidirectional propagation of ultrasound waves, yielding 20 μm resolution at 3 mm depth in turbid mouse brain tissues in vivo.