Endoscopic MPM objective designed for depth scanning

One major advantage of multiphoton microscopy (MPM) is that it can image below tissue surface and produce a stack of images showing sample structure at various depths. A miniature objective with depth scanning capability is needed for MPM endoscopy. Spherical aberration may be induced when changing the focusing depth during multiphoton microscope depth scanning, thus limiting the range over which images may be acquired. A specially designed miniature objective that minimizes spherical aberration across large range of focusing depths is presented. Simulations show that the 0.53 numerical aperture design can achieve on-axis diffraction limited focusing in water for depths from 0 μm to just over 1400 μm and a diffraction limited field of view of up to 290 μm for a 790 nm laser. In experiment, our multiphoton microscope demonstrates a field of view of 64 μm by 100 μm and a depth scanning range of 440 μm, limited by the scanning hardware. Depth scanning capability is confirmed by imaging 0.1 μm diameter fluorescent beads across the 440 μm range. Biological samples to a depth of 150 μm are imaged using the custom objective; the imaging depth is mainly limited by the absorption and scattering of the sample.