Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult brains.

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision.

SOLEIL: single-objective lens inclined light sheet localization microscopy.

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples.

Extended scene deep learning wavefront sensing.

We have applied a combination of blind deconvolution and deep learning to the processing of Shack-Hartmann images. By using the intensity information contained in spot positions, and the fine structure of the separate images created by the lenslets, we have increased the sensitivity and resolution of the sensor over the limit defined by standard processing of spot displacements only. We also have demonstrated the applicability of the method to wavefront sensing using extended objects as a reference.

Phase retrieval of large-scale time-varying aberrations using a non-linear Kalman filtering framework.

This paper presents a computationally efficient framework in which a single focal-plane image is used to obtain a high-resolution reconstruction of dynamic aberrations. Assuming small-phase aberrations, a non-linear Kalman filter implementation is developed whose computational complexity scales close to linearly with the number of pixels of the focal-plane camera. The performance of the method is tested in a simulation of an adaptive optics system, where the small-phase assumption is enforced by considering a closed-loop system that uses a low-resolution wavefront sensor to control a deformable mirror.

Anisoplanatic adaptive optics in parallelized laser scanning microscopy.

Inhomogeneities in the refractive index of a biological microscopy sample can introduce phase aberrations, severely impairing the quality of images. Adaptive optics can be employed to correct for phase aberrations and improve image quality. However, conventional adaptive optics can only correct a single phase aberration for the whole field of view (isoplanatic correction) while, due to the highly heterogeneous nature of biological tissues, the sample induced aberrations in microscopy often vary throughout the field of view (anisoplanatic aberration), limiting significantly the effectiveness of adaptive optics.

Phase retrieval based on the vectorial model of point spread function.

We present an efficient phase retrieval approach for imaging systems with high numerical aperture based on the vectorial model of the point spread function. The algorithm is in the class of alternating minimization methods and can be adjusted for applications with either known or unknown amplitude of the field in the pupil. The algorithm outperforms existing solutions for high-numerical-aperture phase retrieval: (1) the generalization of the method of Hanser et al.

Hartmann-Shack wavefront reconstruction with bitmap image processing.

In this Letter, we report on an algorithm and its implementation to reconstruct the wavefront as a continuous function from a bitmap image of the Hartmann-Shack pattern. The approach works with arbitrary raster geometry and does not require explicit spot definition and phase unwrapping. The system matrix, defining the coefficients of wavefront decomposition in the system of basis functions, is obtained as a result of a series of convolutions and thresholding operations on the reference and sample images.

Predictive wavefront sensorless adaptive optics for time-varying aberrations.

A new wavefront sensorless adaptive optics method is presented that can accurately correct for time-varying aberrations using a single focal plane image at each sample instance. The linear relation between the mean square of the aberration gradient and the change in second moment of the image forms the basis of the presented method. The new algorithm results in significant improvements when an accurate model of the aberration's temporal dynamics is known, by applying a Kalman filter and optimal control.

Fast Calculation of Computer Generated Holograms for 3D Photostimulation through Compressive-Sensing Gerchberg-Saxton Algorithm.

The use of spatial light modulators to project computer generated holograms is a common strategy for optogenetic stimulation of multiple structures of interest within a three-dimensional volume. A common requirement when addressing multiple targets sparsely distributed in three dimensions is the generation of a points cloud, focusing excitation light in multiple diffraction-limited locations throughout the sample. Calculation of this type of holograms is most commonly performed with either the high-speed, low-performance random superposition algorithm, or the low-speed, high performance Gerchberg-Saxton algorithm.

Extended-image-based correction of aberrations using a deformable mirror with hysteresis.

With a view to the next generation of large space telescopes, we investigate guide-star-free, image-based aberration correction using a unimorph deformable mirror in a plane conjugate to the primary mirror. We designed and built a high-resolution imaging testbed to evaluate control algorithms. In this paper we use an algorithm based on the heuristic hill climbing technique and compare the correction in three different domains, namely the voltage domain, the domain of the Zernike modes, and the domain of the singular modes of the deformable mirror.

Pupil mask diversity for image correction in microscopy.

Three-dimensional microscopy suffers from sample-induced aberrations that reduce the resolution and lead to misinterpretations of the object distribution. In this paper, the resolution of a three-dimensional fluorescent microscope is significantly improved by introducing an amplitude diversity in the form of a binary amplitude mask positioned in several different orientations within the pupil, followed by computer processing of the diversity images. The method has proved to be fast, easy to implement, and cost-effective in high-resolution imaging of casper fli:GFP zebrafish.

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications.

Optical path difference microscopy with a Shack-Hartmann wavefront sensor.

In this Letter, we show that a Shack-Hartmann wavefront sensor can be used for the quantitative measurement of the specimen optical path difference (OPD) in an ordinary incoherent optical microscope, if the spatial coherence of the illumination light in the plane of the specimen is larger than the microscope resolution. To satisfy this condition, the illumination numerical aperture should be smaller than the numerical aperture of the imaging lens. This principle has been successfully applied to build a high-resolution reference-free instrument for the characterization of the OPD of micro-optical components and microscopic biological samples.

Adaptive illumination based on direct wavefront sensing in a light-sheet fluorescence microscope.

A methodology for the adaptive control and correction of phase aberrations in the illumination arm of a light-sheet fluorescence microscope has been developed. The method uses direct wavefront sensing on epi-fluorescent light to detect the aberration present in the sample. Using this signal, the aberrations in the illumination arm are subsequently corrected with a spatial light modulator in a feedforward mode.

Holographic imaging with a Shack-Hartmann wavefront sensor.

A high-resolution Shack-Hartmann wavefront sensor has been used for coherent holographic imaging, by computer reconstruction and propagation of the complex field in a lensless imaging setup. The resolution of the images obtained with the experimental data is in a good agreement with the diffraction theory. Although a proper calibration with a reference beam improves the image quality, the method has a potential for reference-less holographic imaging with spatially coherent monochromatic and narrowband polychromatic sources in microscopy and imaging through turbulence.

Pupil filters for extending the field-of-view in light-sheet microscopy.

Pupil filters, represented by binary phase modulation, have been applied to extend the field of view of a light-sheet fluorescence microscope. Optimization has been used, first numerically to calculate the optimum filter structure and then experimentally, to scale and align the numerically synthesized filter in the microscope. A significant practical extension of the field of view has been observed, making the reported approach a valuable tool on the path to wide-field light-sheet microscopy.

Model-based wavefront sensorless adaptive optics system for large aberrations and extended objects.

A model-based wavefront sensorless (WFSless) adaptive optics (AO) system with a 61-element deformable mirror is simulated to correct the imaging of a turbulence-degraded extended object. A fast closed-loop control algorithm, which is based on the linear relation between the mean square of the aberration gradients and the second moment of the image intensity distribution, is used to generate the control signals for the actuators of the deformable mirror (DM). The restoration capability and the convergence rate of the AO system are investigated with different turbulence strength wave-front aberrations.

Plenoptic wavefront sensor with scattering pupil.

We consider a wavefront sensor combining scattering pupil with a plenoptic imager. Such a sensor utilizes the same reconstruction principle as the Hartmann-Shack sensor, however it is free from the ambiguity of the spot location caused by the periodic structure of the sensor matrix, and allows for wider range of measured aberrations. In our study, sensor with scattering pupil has demonstrated a good match between the introduced and reconstructed aberrations, both in the simulation and experiment.

Speckle imaging through turbulent atmosphere based on adaptable pupil segmentation.

We report on the first results to our knowledge obtained with adaptable multiaperture imaging through turbulence on a horizontal atmospheric path. We show that the resolution can be improved by adaptively matching the size of the subaperture to the characteristic size of the turbulence. Further improvement is achieved by the deconvolution of a number of subimages registered simultaneously through multiple subapertures.

Correction of low order aberrations using continuous deformable mirrors.

By analyzing the Poisson equation describing the static behavior of membrane and bimorph deformable mirrors and biharmonic equation describing the continuous facesheet mirror with push-pull actuators, we found that to achieve a high quality correction of low-order aberrations these mirrors should have sufficient number of actuators positioned outside the correction aperture. In particular, any deformable mirror described by the Poisson equation requires at least two actuators to be placed outside the working aperture per period of the azimuthal aberration of the highest expected order. Any deformable mirror described by the biharmonic equation, such as a continuous facesheet mirror with push-pull actuators, requires at least four actuators to be placed outside the working aperture per period of the azimuthal aberration of the highest expected order, and these actuators should not be positioned on a single circle.

Hartmann-Shack test with random masks for modal wavefront reconstruction.

The paper discusses the influence of the geometry of a Hartmann-(Shack) wavefront sensor on the total error of modal wavefront reconstruction. A mathematical model is proposed, which describes the modal wavefront reconstruction in terms of linear operators. The model covers the most general case and is not limited by the orthogonality of decomposition basis or by the method chosen for decomposition.

Phase extraction from three and more interferograms registered with different unknown wavefront tilts.

We propose phase retrieval from three or more interferograms corresponding to different tilts of an object wavefront. The algorithm uses the information contained in the interferogram differences to reduce the problem to phase shifting. Three interferograms is the minimum for restoring the phase over most of the image.