Large depth-of-field ultraviolet-visible photoacoustic histologic and microvascular imaging in vivo

Photoacoustic microscopy (PAM) can image diverse biological microstructures by harnessing characteristic optical absorption spectra of intrinsic nonfluorescent biomolecules. We incorporate ultraviolet (UV) and visible pulsed lasers into a PAM to develop an UV-visible PAM for label-free histologic and microvascular imaging, where the cell nuclei and blood vessels are specifically captured at high contrast relying on strong optical absorption of DNA/RNA and hemoglobin at 266 and 532 nm wavelengths, respectively. Moreover, two diffractive optical elements operating at UV and visible spectra are designed for engineering the excitation beams, significantly enlarging depths of field (DOFs > 200 μm). The UV-visible PAM demonstrates combined capabilities of dual imaging contrast, elongated DOFs, and micrometer-scale lateral resolution for delineating the spatial microarchitectures of both cell nuclei and blood vessels that are at different depth locations in the biological specimens with uneven surfaces. Longitudinal monitoring of the trauma is performed in mouse ear in vivo. Potentially, our UV-visible PAM could offer comprehensive histologic and microvascular information in a broad range of biomedical studies.