Optical aberrations due to the inhomogeneous refractive index of tissue degrade the resolution and brightness of images in deep-tissue imaging. We introduce a confocal fluorescence microscope with adaptive optics, which can correct aberrations based on direct wavefront measurements using a Shack-Hartmann wavefront sensor with a fluorescent bead used as a point source reference beacon. The results show a 4.3× improvement in the Strehl ratio and a 240% improvement in the signal intensity for fixed mouse tissues at depths of up to 100 μm.

This presentation will be on the use of adaptive optics (AO) with direct wavefront sensing for biological imaging. Adaptive optics have been used in ground based astronomy to correct image aberrations caused by refraction as light passes through Earth's turbulent atmosphere. As shown on the left in Figure One, light from the telescope has a distorted wavefront, as indicated by the wavy lines. A wavefront sensor measures these distortions and applies the opposite shape on an adaptive mirror using a feedback control system. After reflection from the adaptive mirror a corrected wavefront is generated and is recorded by a highresolution camera. An image of the planet Neptune before and after AO correction is shown on the right in Figures 1(a) and 1(b). After correction the cloud structure on Neptune can be resolved in 1(b)

We present a new method to directly measure and correct the aberrations introduced when imaging through thick biological tissue. A Shack-Hartmann wavefront sensor is used to directly measure the wavefront error induced by a Drosophila embryo. The wavefront measurements are taken by seeding the embryo with fluorescent microspheres used as “artificial guide-stars.” The wavefront error is corrected in ten millisecond steps by applying the inverse to the wavefront error on a micro-electro-mechanical deformable mirror in the image path of the microscope. The results show that this new approach is capable of improving the Strehl ratio by 2 times on average and as high as 10 times when imaging through 100 μm of tissue. The results also show that the isoplanatic half-width is approximately 19 μm resulting in a corrected field of view 38 μm in diameter around the guide-star.

Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy.

We report a technique for measuring and correcting the wavefront aberrations introduced by a biological sample using a Shack-Hartmann wavefront sensor, a fluorescent reference source, and a deformable mirror. The reference source and sample fluorescence are at different wavelengths to separate wavefront measurement and sample imaging. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved.