Miniature objective lens design for multiphoton microscopy

Multiphoton microscopy is a promising imaging technique capable of optical sectioning to acquire images from below the surface of tissue. A miniature objective with depth-scanning capability is needed for clinically adaptation. Imaging performance is dependent upon the ability to focus pulsed laser light. State-of-the-art endoscopes have a depth-scanning capability of ~200 μm and suffer from a significant decrease in resolution at the limit of their depth-scanning range. Zemax software is used to evaluate aspherical lenses for use as objectives, to evaluate the effect of scanning optics, and to design a custom water-dipping objective. Simulations show that the custom objective is capable of achieving diffraction-limited focusing for depths from 0 μm to 1400 μm in water by minimizing spherical aberration. A shape memory alloy actuator is used by the objective for depth-scanning across a 440 μm range, which is experimentally demonstrated by imaging fluorescent beads across the entire range and is more than double the range capability of MPM endoscopes in literature. Resolution measured ~0.7 μm laterally and ~10 μm axially and remained consistent across the entire depth-scanning range. This is a significant improvement to both our lab’s imaging ability and to the depth-scanning abilities of endoscopes presented in literature. Resolution is improved by the addition of corrective optics that reduces axial chromatic aberration. The effect of chromatic aberration on the focusing ability of the custom objective is investigated via simulation using Zemax. Simulations show that the 3 dB wavelengths of the laser’s 80 fs pulses focus at a different depth than the central wavelength and also that marginal rays and on-axis rays arrive at the focal point at different times. By adding CO to reduce chromatic aberration, both of these differences are significantly reduced, which improves the signal to noise ratio. An equation to calculate the difference in arrival time is derived, which shows that the difference is proportional to the square of the objective’s numerical aperture. The deep imaging capabilities of the water-dipping miniature MPM objective is demonstrated by imaging tissue and shows great promise for future clinical use.Applied Science, Faculty ofElectrical and Computer Engineering, Department ofGraduat