Reflection-mode submicron-resolution in vivo photoacoustic microscopy.

Submicron-resolution photoacoustic microscopy (PAM) currently exists only in transmission mode, due to the technical difficulties of combining high numerical-aperture (NA) optical illumination with high NA acoustic detection. The lateral resolution of reflection-mode PAM has not reached <2 μm in the visible light range. Here we develop the first reflection-mode submicron-resolution PAM system with a new compact design. By using a parabolic mirror to focus and reflect the photoacoustic waves, sufficient signals were collected for good sensitivity without distorting the optical focusing. By imaging nanospheres and a resolution test chart, the lateral resolution was measured to be ≈ 0.5 μm with an optical wavelength of 532 nm, an optical NA of 0.63. The axial resolution was measured at 15 μm. Here the axial resolution was measured by a different experiment with the lateral resolution measurement. But we didn't describe the details of axial resolution measurement due to space limit. The maximum penetration was measured at ≈ 0.42 mm in optical-scattering soft tissue. As a comparison, both the submicron-resolution PAM and a 2.4 μm-resolution PAM were used to image a mouse ear in vivo with the same optical wavelength and similar pulse energy. Capillaries were resolved better by the submicron-resolution PAM. Therefore, the submicron-resolution PAM is suitable for in vivo high-resolution imaging, or even subcellular imaging, of optical absorption.