A biopsy-needle compatible varifocal multiphoton rigid probe for depth-resolved optical biopsy

In this work, we report a biopsy-needle compatible rigid probe, capable of performing three-dimensional (3D) two-photon optical biopsy. The probe has a small outer diameter of 1.75 mm and fits inside a gauge-14 biopsy needle to reach internal organs. A carefully designed focus scanning mechanism has been implemented in the rigid probe, which, along with a rapid two-dimensional MEMS scanner, enables 3D imaging. Fast image acquisition up to 10 frames per second is possible, dramatically reducing motion artifacts during in vivo imaging. Equipped with a high-numerical aperture micro-objective, the miniature rigid probe offers a high two-photon resolution (0.833 x 6.11 mu m, lateral x axial), a lateral field of view of 120 mu m, and an axial focus tuning range of 200 mu m. In addition to imaging of mouse internal organs and subcutaneous tumor in vivo, first-of-its-kind depth-resolved two-photon optical biopsy of an internal organ has been successfully demonstrated on mouse kidney in vivo and in situ.