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. This paper reports on a new approach for aberration correction in laser scanning confocal microscopy, in which a spatial light modulator is used to generate multiple excitation points in the sample to simultaneously scan different portions of the field of view with completely independent correction, achieving anisoplanatic compensation of sample induced aberrations, in a significantly shorter time compared to sequential isoplanatic correction of multiple image subregions. The method was tested in whole Drosophila brains and in larval Zebrafish, each showing a dramatic improvement in resolution and sharpness when compared to conventional isoplanatic adaptive optics.

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In this Letter, we present a solution for simple implementation of adaptive optics in any existing laser scanning fluorescence microscope. Adaptive optics are implemented by the introduction of a multiactuator adaptive lens between the microscope body and the objective lens. Correction is performed with a sensorless method by optimizing the quality of the images presented on screen by the microscope software. We present the results acquired on both a commercial linear excitation confocal microscope and a custom-made multiphoton excitation microscope.

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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. This paper presents a variation of the Gerchberg–Saxton algorithm that, by only performing iterations on a subset of the data, according to compressive sensing principles, is rendered significantly faster while maintaining high quality outputs. The algorithm is presented in high-efficiency and high-uniformity variants. All source code for the method implementation is available as Supplementary Materials and as open-source software. The method was tested computationally against existing algorithms, and the results were confirmed experimentally on a custom setup for in-vivo multiphoton optogenetics. The results clearly show that the proposed method can achieve computational speed performances close to the random superposition algorithm, while retaining the high performance of the Gerchberg–Saxton algorithm, with a minimal hologram quality loss@en

In this work, we present a new confocal laser scanning microscope capable to perform sensorless wavefront optimization in real time. The device is a parallelized laser scanning microscope in which the excitation light is structured in a lattice of spots by a spatial light modulator, while a deformable mirror provides aberration correction and scanning. A binary DMD is positioned in an image plane of the detection optical path, acting as a dynamic array of reflective confocal pinholes, images by a high performance CMOS camera. A second camera detects images of the light rejected by the pinholes for sensorless aberration correction.@en

In wavefront characterization, often the combination of a Shack-Hartmann sensor and a reconstruction method utilizing the Cartesian derivatives of Zernike circle polynomials (the least-squares method, to be called here Method A) is used, which is known to introduce crosstalk. In [J. Opt. Soc. Am. A 31, 1604 (2014), a crosstalk-free analytic expression of the LMS estimator of the wavefront Zersectnike coefficients is given in terms of wavefront partial derivatives (leading to what we call Method B). Here, we show an implementation of this analytic result where the derivative data are obtained using the Shack-Hartmann sensor and compare it with the conventional least-squares method.@en

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. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

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In life sciences, interest in the microscopic imaging of increasingly complex three dimensional samples, such as cell spheroids, zebrafish embryos, and in vivo applications in small animals, is growing quickly. Due to the increasing complexity of samples, more and more life scientists are considering the implementation of adaptive optics in their experimental setups. While several approaches to adaptive optics in microscopy have been reported, it is often difficult and confusing for the microscopist to choose from the array of techniques and equipment. In this poster presentation we offer a small guide to adaptive optics providing general guidelines for successful adaptive optics implementation.

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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 di erent orientations within the pupil, followed by computer processing of the diversity images. The method has proved to be fast, easy to implement, and cost-e ective in high-resolution imaging of casper fli:GFP zebrafish.@en

Adaptive Optics (AO) has revealed as a very promising technique for high-resolution microscopy, where the presence of optical aberrations can easily compromise the image quality. Typical AO systems however, are almost impossible to implement on commercial microscopes. We propose a simple approach by using a Multi-actuator Adaptive Lens (MAL) that can be inserted right after the objective and works in conjunction with an image optimization software allowing for a wavefront sensorless correction. We presented the results obtained on several commercial microscopes among which a confocal microscope, a fluorescence microscope, a light sheet microscope and a multiphoton microscope.@en

The light-sheet fluorescence microscopy is an excellent tool for the investigation of large three dimensional microscopy samples at the cellular level, however, the ability to resolve features is strongly affected by the presence of scattering and aberrations. These effects are two fold in light-sheet microscopy, as the illumination path providing the optical sectioning and the fluorescence detection path are both affected by the aberrations in different ways. To overcome these difficulties, we have developed hybrid adaptive optical and computational microscopy techniques to remove the effect of the aberrations in both the excitation and the fluorescence paths of these microscopes.@en

The quality of fluorescence microscopy images is often impaired by the presence
of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The
paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.@en

A methodology for retrieving the unknown object distribution and point-spread functions (PSFs) from a set of images acquired in the presence of temporal phase aberrations is presented in this paper. The method works by finding optimal complimentary linear filters for multi-frame deconvolution. The algorithm uses undemanding computational operations and few a priori, making it simple, fast and robust even at low signal-to-noise ratios. Results of numerical simulations and experimental tests are given as empirical proof, alongside comparisons with other algorithms found in the literature.

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By taking multiple input-output measurements, it is shown how to determine the input to an optical system that corrects unknown phase aberrations without interferometric measurements or online iterative optimization within a couple of seconds. It is shown to work in simulations and experiment. This technique may also be used to acquire the complex field in the pupil, hereby permitting a complex field image to be acquired. @en

Light-sheet fluorescence microscopy techniques commonly use deconvolution to remove the effect of the illumination beam shape on the image formation, reducing the effects of side lobes and increasing the contrast in three-dimensional imaging. Deconvolution requires knowledge of the optical transfer function of the system and can only be estimated or measured imperfectly. Furthermore, in biological samples the optical transfer function is degraded by the presence of phase aberrations, this implies that any a priori deconvolution applied to the sample will be to some degree incorrect since it does not account for this unknown phase error in the optical transfer function. By combining adaptive optics and computational processing in this microscope, it is shown that by introducing perturbations to the optical transfer function it is possible to estimate the object distribution and remove aberrations.

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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.

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The use of adaptive lenses instead of deformable mirrors can simplify the implementation of an adaptive optics system. The recently introduced Multi-actuator Adaptive Lens (MAL) can be used in closed loop with a wavefront sensor to correct for time-variant wavefront aberrations. The MAL can guarantee a level of correction and a response time similar to the ones obtained with deformable mirrors. The adaptive lens is based on the use of piezoelectric actuators and, without any obstruction or electrodes in the clear aperture, can guarantee a fast response time, less than ∼10ms. Our tests show that the MAL can be used both in combination with a wavefront sensor in a "classical" adaptive optics closed loop, or in a wavefront sensorless configuration. The latter has allowed us to design more compact and simple imaging systems for different microscopy platforms. We will show that the Multi-actuator Adaptive Lens has been successfully used for in-vivo OCT ophthalmic imaging in both mice and humans, as well as confocal and two photon microscopy. We tested and compared different optimization strategies such as coordinate search and the DONE algorithm. The results suggest that the MAL optimization can correct for eye aberrations with a pupil of 5mm or sample induced aberrations in microscopy.@en

We present a new method for fast wavefront sensorless correction of phase aberrations in confocal microscopy, based on a physical model of the distribution of fluorescence light rejected by the pinhole. The method is described, and an experimental confirmation of the method is provided.@en

This proceeding reports early results in the development of a new technique for adaptive optics in confocal microscopy. The term adaptive optics refers to the branch of optics in which an active element in the optical system is used to correct inhomogeneities in the media through which light propagates. In its most classical form, mostly used in astronomical imaging, adaptive optics is achieved through a closed loop in which the actuators of a deformable mirror are driven by a wavefront sensor. This approach is severely limited in fluorescence microscopy, as the use of a wavefront sensor requires the presence of a bright, point like source in the field of view, a condition rarely satisfied in microscopy samples. Previously reported approaches to adaptive optics in fluorescence microscopy are therefore limited to the inclusion of fluorescent microspheres in the sample, to use as bright stars for wavefront sensors, or time consuming sensorless optimization procedures, requiring several seconds of optimization before the acquisition of a single image. We propose an alternative approach to the problem, implementing sensorless adaptive optics in a Programmable array microscope. A programmable array microscope is a microscope based on a digital micromirror device, in which the single elements of the micromirror act both as point sources and pinholes.@en

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.@en