A fast direct wavefront sensing method for dynamic in-vivo adaptive optical two photon microscopy has demonstrated.
By using the direct wavefront sensing and open loop control, the system provides high-speed wavefront measurement
and correction. To measure the wavefront in the middle of a Drosophila embryo at early stages, autofluorescence from
endogenous fluorophores in the yolk were used as reference guide-stars. This method does not rely on
fluorescently labeled proteins as guide-stars, which can simplify the sample preparation for wavefront measurement. The
method was tested through live imaging of a Drosophila embryo. The aberration in the middle of the embryo was
measured directly for the first time. After correction, both contrast and signal intensity of the structure in the middle of
the embryo was improved.
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.