Development and testing of an AO-structured illumination microscope

The design of an Adaptive Optics (AO) Structured Illumination (SI) microscope is presented. Two key technologies are combined to provide effective super-resolution at significant depths in tissue. AO is used to measure and compensate for optical aberrations in both the system and the tissue by measuring the optical path differences in the wavefront. Uncorrected, these aberrations significantly reduce imaging resolution, particularly as we view deeper into tissue. SI allows us to reconstruct an image with resolution beyond the Rayleigh limit of the optics by aliasing high spatial frequencies, outside the limit of the optics, to lower frequencies within the system pass band. The aliasing is accomplished by spatially modulating the illumination at a frequency near the cutoff frequency of the system. These aliased frequencies are superimposed on the lower spatial frequencies of the object in our image. Using multiple images and an inverse algorithm, we separate the aliased and normal frequencies, restore them to their original frequency positions, and recreate the original spectrum of the object. This allows us to recreate a super-resolution image of the object. A problem arises with thick aberrating tissue. Tissue aberrations, including sphere, increase with depth into the tissue and reduce the high spatial frequency response of a system. This degrades the ability of SI to reconstruct at superresolution and limits its use to relatively shallow depths. However, adding AO to the system compensates for these aberrations allowing SI to work at maximum efficiency even deep within aberrating tissue.