The small correction volume for conventional wavefront shaping methods limits their application in biological imaging through scattering media. In this paper, we take advantage of conjugate adaptive optics (CAO) and remote focusing (CAORF) to achieve three-dimensional (3D) scanning through a scattering layer with a single correction. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We demonstrate two-photon imaging with CAORF through mouse skull. The fluorescent microspheres embedded under the scattering layers can be clearly observed after applying the correction.

A woofer–tweeter adaptive optical structured illumination microscope (AOSIM) is presented. By combining a low-spatial-frequency large-stroke deformable mirror (woofer) with a high-spatial-frequency low-stroke deformable mirror (tweeter), we are able to remove both large-amplitude and high-order aberrations. In addition, using the structured illumination method, as compared to widefield microscopy, the AOSIM can accomplish high-resolution imaging and possesses better sectioning capability. The AOSIM was tested by correcting a large aberration from a trial lens in the conjugate plane of the microscope objective aperture. The experimental results show that the AOSIM has a point spread function with an FWHM that is 140 nm wide (using a water immersion objective lens with NA=1.1) after correcting a large aberration (5.9 μm peak-to-valley wavefront error with 2.05 μm RMS aberration). After structured light illumination is applied, the results show that we are able to resolve two beads that are separated by 145 nm, 1.62× below the diffraction limit of 235 nm. Furthermore, we demonstrate the application of the AOSIM in the field of bioimaging. The sample under investigation was a green-fluorescent-protein-labeled Drosophila embryo. The aberrations from the refractive index mismatch between the microscope objective, the immersion fluid, the cover slip, and the sample itself are well corrected. Using AOSIM we were able to increase the SNR for our Drosophila embryo sample by 5×.

The use of adaptive optics correction for structured illumination microscopy requires spatial sinusoid patterns in the sample obtained via two beam interference. We simulate the effect of AO correction on both excitation and emission paths, and propose a better SIM microscope based that considers the special requirements for SIM.

The small correction volume for conventional wavefront shaping methods limits their applications in biological imaging through scattering media. We demonstrate large volume wavefront shaping through a scattering layer with a single correction by conjugate adaptive optics and remote focusing (CAORF). The remote focusing module can keep the conjugation between the AO and scattering layer during three-dimensional scanning. This new configuration provides a wider correction volume by the best utilization of the memory effect in a fast three-dimensional laser scanning microscope. Our results show that the proposed system can provide 10 times wider axial field of view compared with a conventional conjugate AO system when 16,384 segments are used on a spatial light modulator. We also demonstrated three-dimensional fluorescence imaging and multi-spot patterning through a scattering layer.

In this work, we propose a structured illumination (SI) method based on a two-photon excitation (TPE) scanning laser beam. Advantages of TPE methods include optical sectioning, low phototoxicity, and robustness in the face of sample-induced scattering. We designed a novel multispot point spread function (PSF) for a fast, two-photon scanning SIM microscope. Our multispot PSF is generated with a phase retrieval algorithm. We show how to obtain the phase distribution and then simulate the effect of this distribution on a spatial light modulator (SLM), which produces the multispot PSF in the object plane of the microscope. We produce simulations that show the viability of this method. The results are simulated and a multispot PSF scanning SIM microscope is proposed.

Our ability to see fine detail at depth in tissues is limited by scattering and other refractive characteristics of the tissue. For fixed tissue, we can limit scattering with a variety of clearing protocols. This allows us to see deeper but not necessarily clearer. Refractive aberrations caused by the bulk index of refraction of the tissue and its variations continue to limit our ability to see fine detail. Refractive aberrations are made up of spherical and other Zernike modes, which can be significant at depth. Spherical aberration that is common across the imaging field can be corrected using an objective correcting collar, although this can require manual intervention. Other aberrations may vary across the imaging field and can only be effectively corrected using adaptive optics. Adaptive optics can also correct other aberrations simultaneously with the spherical aberration, eliminating manual intervention and speeding imaging. We use an adaptive optics two-photon microscope to examine the impact of the spherical and higher order aberrations on imaging and contrast the effect of compensating only for spherical aberration against compensating for the first 22 Zernike aberrations in two tissue types. Increase in image intensity by 1.6× and reduction of root mean square error by 3× are demonstrated.

In this paper, we demonstrate a fast binary intensity modulation based on the measurement of the binary TM. For each correction, the binary TM was calculated based on measurements of the intensity change at the target with a series of input masks. After preloading the measurement masks, the DMD can run at full speed during measurement. The system allows dynamic focusing at 12.5 Hz with 1024 input modes, and more than 60 times intensity enhancement. We demonstrate focusing light through a highly dynamic scattering sample, a live drosophila embryo.

Optical sectioning of biological tissues has become the method of choice for three-dimensional histological analyses. This is particularly important in the brain were neurons can extend processes over large distances and often whole brain tracing of neuronal processes is desirable. To allow deeper optical penetration, which in fixed tissue is limited by scattering and refractive index mismatching, tissue-clearing procedures such as CLARITY have been developed. CLARITY processed brains have a nearly uniform refractive index and three-dimensional reconstructions at cellular resolution have been published. However, when imaging in deep layers at submicron resolution some limitations caused by residual refractive index mismatching become apparent, as the resulting wavefront aberrations distort the microscopic image. The wavefront can be corrected with adaptive optics. Here, we investigate the wavefront aberrations at different depths in CLARITY processed mouse brains and demonstrate the potential of adaptive optics to enable higher resolution and a better signal-to-noise ratio. Our adaptive optics system achieves high-speed measurement and correction of the wavefront with an open-loop control using a wave front sensor and a deformable mirror. Using adaptive optics enhanced microscopy, we demonstrate improved image quality wavefront, point spread function, and signal to noise in the cortex of YFP-H mice.

In this work, we propose a structured illumination (SI) method based on a two-photon excitation (TPE) scanning laser beam. Advantages of TPE methods include optical sectioning, low photo-toxicity, and robustness in the face of sample induced scattering. We designed a novel multi-spot point spread function (PSF) for a fast, two-photon scanning SIM microscope. Our multi-spot PSF is generated with a phase retrieval algorithm. We show how to obtain the phase distribution and then simulate the effect of this distribution on a spatial light modulator (SLM), which produces the multi-spot PSF in the object plane of the microscope. We produce simulations that show the viability of this method. The results are simulated and a multi-spot PSF scanning SIM microscope is proposed.

Using the fast measurement of a binary transmission matrix and a digital micromirror device, we demonstrate high-speed interferometric focusing through highly dynamic scattering media with binary intensity modulation. The scanning of speckles for reference optimization gives stable focusing, which can be used for focusing through a fast changing media or two dimensional scanning through a slowly changing scattering media. The system allows dynamic focusing at 12.5 Hz with 1024 input modes, and more than 60 times intensity enhancement. It was tested with a moving diffuser, a mouse brain and skull tissue. The experiment with a live drosophila embryo shows its potential in compensating dynamic scattering in live biological tissue.

The multiple light scattering in biological tissues limits the measurement depth for traditional wavefront sensor. The attenuated ballistic light and the background noise caused by the diffuse light give low signal to noise ratio for wavefront measurement. To overcome this issue, we introduced a wavefront estimation method based on a ray tracing algorithm to overcome this issue. With the knowledge of the refractive index of the medium, the wavefront is estimated by calculating optical path length of rays from the target inside of the samples. This method can provide not only the information of spherical aberration from the refractive-index mismatch between the medium and biological sample but also other aberrations caused by the irregular interface between them. Simulations based on different configurations are demonstrated in this paper.

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.

The Lick Observatory's Shane 3-meter telescope has been upgraded with a new infrared instrument (ShARCS - Shane Adaptive optics infraRed Camera and Spectrograph) and dual-deformable mirror adaptive optics (AO) system (ShaneAO). We present first-light measurements of imaging sensitivity in the Ks band. We compare measured results to predicted signal-to-noise ratio and magnitude limits from modeling the emissivity and throughput of ShaneAO and ShARCS. The model was validated by comparing its results to the Keck telescope adaptive optics system model and then by estimating the sky background and limiting magnitudes for IRCAL, the previous infra-red detector on the Shane telescope, and comparing to measured, published results. We predict that the ShaneAO system will measure lower sky backgrounds and achieve 20% higher throughput across the JHK bands despite having more optical surfaces than the current system. It will enable imaging of fainter objects (by 1-2 magnitudes) and will be faster to reach a fiducial signal-to-noise ratio by a factor of 10-13. We highlight the improvements in performance over the previous AO system and its camera, IRCAL.

We evaluate the performance of a woofer-tweeter controller architecture for the new 3-meter Shane Telescope (Lick Observatory) laser guidestar adaptive optics (AO) system. Low order, high stroke phase correction is performed using the normal modal basis set of the Alpao woofer deformable mirror (DM). Since the woofer and tweeter DMs share the same wavefront sensor, the projected woofer phase correction is offloaded from the high-order, low stroke phase aberrations corrected by the tweeter DM. This ensures the deformable mirrors complementarily correct the input phase disturbance and minimizes likelihood of the tweeter actuators saturating. Preliminary analysis of on-sky closed-loop deformable mirror telemetry data from currently operating AO systems at Mt. Hamilton, as well as statistically accurate Kolmogorov phase screens, indicate that correction of up to 34 woofer modes results in all tweeter actuators remaining within their stroke limit.

The Lick Observatory 3-meter telescope has a history of serving as a testbed for innovative adaptive optics techniques. In 1996, it became one of the first astronomical observatories to employ laser guide star (LGS) adaptive optics as a facility instrument available to the astronomy community. Work on a second-generation LGS adaptive optics system, ShaneAO, is well underway, with plans to deploy on telescope in 2013. In this paper we discuss key design features and implementation plans for the ShaneAO adaptive optics system. Once again, the Shane 3-m will host a number of new techniques and technologies vital to the development of future adaptive optics systems on larger telescopes. Included is a woofer-tweeter based wavefront correction system incorporating a voice-coil actuated, low spatial and temporal bandwidth, high stroke deformable mirror in conjunction with a high order, high bandwidth MEMs deformable mirror. The existing dye laser, in operation since 1996, will be replaced with a fiber laser recently developed at Lawrence Livermore National Laboratories. The system will also incorporate a high-sensitivity, high bandwidth wavefront sensor camera. Enhanced IR performance will be achieved by replacing the existing PICNIC infrared array with an Hawaii 2RG. The updated ShaneAO system will provide opportunities to test predictive control algorithms for adaptive optics. Capabilities for astronomical spectroscopy, polarimetry, and visible-light adaptive optical astronomy will be supported.

Micro-electro-mechanical systems (MEMS) technology can provide for deformable mirrors (DMs) with excellent performance within a favorable economy of scale. Large MEMS-based astronomical adaptive optics (AO) systems such as the Gemini Planet Imager are coming on-line soon. As MEMS DM end-users, we discuss our decade of practice with the micromirrors, from inspecting and characterizing devices to evaluating their performance in the lab. We also show MEMS wavefront correction on-sky with the "Villages" AO system on a 1-m telescope, including open-loop control and visible-light imaging. Our work demonstrates the maturity of MEMS technology for astronomical adaptive optics.

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.

We demonstrated the used of an adaptive optic system in biological imaging to improve the imaging characteristics of a wide field microscope. A crimson red fluorescent bead emitting light at 650 nm was used together with a Shack-Hartmann wavefront sensor and deformable mirror to compensate for the aberrations introduce by a Drosophila embryo. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved (510 nm). The use of the crimson beads allow for less photobleaching to be done to the science object of the embryo, in this case our GFP model (green fluorescent beads), and allows for the science object and wavefront reference to be spectrally separated. The spectral separation allows for single points sources to be used for wavefront measurements, which is a necessary condition for the Shack-Hartmann Wavefront sensor operation.

The Keck Next Generation Adaptive Optics (KNGAO) system promises to yield high-Strehl observations over a wide range of science wavelengths from the optical through the infrared. We describe the algorithms proposed for a Real-Time Controller (RTC) implemented in a massive parallel processor environment. These algorithms take advantage of the Fourier domain to speed up processing and ensure minimum variance control that incorporates prior as well as current data. We present the unique approach to the design that enables such a complex tomography processor to scale more favorably with telescope aperture size than the more traditional RTC approaches.

At the University of California's Lick Observatory, we have implemented an on-sky testbed for next-generation adaptive optics (AO) technologies. The Visible-Light Laser Guidestar Experiments instrument (ViLLaGEs) includes visible-light AO, a micro-electro-mechanical-systems (MEMS) deformable mirror, and open-loop control of said MEMS on the 1-meter Nickel telescope at Mt. Hamilton. In this paper we evaluate the performance of ViLLaGEs in open- and closed-loop control, finding that both control methods give equivalent Strehl ratios of up to similar to 7% in I-band and similar rejection of temporal power. Therefore, we find that open- loop control of MEMS on-sky is as effective as closed-loop control. Furthermore, after operating the system for three years, we find MEMS technology to function well in the observatory environment. We construct an error budget for the system, accounting for 130 nm of wavefront error out of 190 nm error in the science-camera PSFs. We find that the dominant known term is internal static error, and that the known contributions to the error budget from open- loop control (MEMS model, position repeatability, hysteresis, and WFS linearity) are negligible.