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.

Large volume holographic imaging for biological sample analysis.

SIGNIFICANCE: Particle field holography is a versatile technique to determine the size and distribution of moving or stationary particles in air or in a liquid without significant disturbance of the sample volume. Although this technique is applied in biological sample analysis, it is limited to small sample volumes, thus increasing the number of measurements per sample. In this work, we characterize the maximum achievable volume limit based on the specification of a given sensor to realize the development of a potentially low-cost, single-shot, large-volume holographic microscope. AIM: We present mathematical formulas that will aid in the design and development and improve the focusing speed for the numerical reconstruction of registered holograms in particle field holographic microscopes. Our proposed methodology has potential application in the detection of Schistosoma haematobium eggs in human urine samples. APPROACH: Using the Fraunhofer holography theory for opaque objects, we derived an exact formula for the maximum diffraction-limited volume for an in-line holographic setup. The proof-of-concept device built based on the derived formulas was experimentally validated with urine spiked with cultured Schistosoma haematobium eggs. RESULTS: Results obtained show that for urine spiked with Schistosoma haematobium eggs, the volume thickness is limited to several millimeters due to scattering properties of the sample. The distances of the target particles could be estimated directly from the hologram fringes. CONCLUSION: The methodology proposed will aid in the development of large-volume holographic microscopes.

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. 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.

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.

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. Through our systematic experimental study, we found that successive control in two domains effectively counteracts uncompensated hysteresis of the deformable mirror.

Imaging & identification of malaria parasites using cellphone microscope with a ball lens.

We have optimized the design and imaging procedures, to clearly resolve the malaria parasite in Giemsa-stained thin blood smears, using simple low-cost cellphone-based microscopy with oil immersion. The microscope uses a glass ball as the objective and the phone camera as the tube lens. Our optimization includes the optimal choice of the ball lens diameter, the size and the position of the aperture diaphragm, and proper application of immersion, to achieve diagnostic capacity in a wide field of view. The resulting system is potentially applicable to low-cost in-the-field optical diagnostics of malaria as it clearly resolves micron-sized features and allows for analysis of parasite morphology in the field of 50 × 50 µm, and parasite detection in the field of at least 150 × 150 µm.

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

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. 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.

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.

Aliasing, coherence, and resolution in a lensless holographic microscope.

We have shown that the maximum achievable resolution of an in-line lensless holographic microscope is limited by aliasing and, for collimated illumination, cannot exceed the camera pixel size. This limit can be achieved only when the optimal conditions on the spatial and temporal coherence state of the illumination are satisfied. The expressions defining the configuration, delivering maximum resolution with given spatial and temporal coherence of the illumination, are obtained. The validity of these conditions is confirmed experimentally.

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. Adaptive correction, resulting in significant improvement in the axial resolution, has been demonstrated by imaging Tg(fli:GFP) zebrafish embryos.

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.

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. The concept is expected to be applicable to optical metrology of strongly distorted wavefronts, especially for measurements through dirty, distorted, or scattering windows and pupils, such as cataract eyes.

Characterization and closed-loop performance of a liquid mirror adaptive optical system.

A deformable mirror based on the principle of total internal reflection of light from an electrostatically deformed liquid-air interface was realized and used to perform closed-loop adaptive optical (AO) correction on a collimated laser beam aberrated by a rotating phase disk. Equations describing the resonant and oscillatory behavior of the liquid system were obtained and applied to the system under consideration. Characterization of the mirror included open- and closed-loop frequency responses, determination of rise times, the damping times of the liquid, and the influence of liquid surface motion in the absence of external optical aberrations. The performance of the AO system was determined for static and dynamic aberrations for various sets of system parameters. The predictions of the general expressions were compared to the results of the experimental realization and were found to be in good agreement.

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. Different implementations of multiaperture geometry, including pupil multiplication, pupil image sampling, and a plenoptic telescope, are considered. Resolution improvement has been demonstrated on a ∼550 m horizontal turbulent path, using a combination of aperture sampling, speckle image processing, and, optionally, frame selection.

Closed-loop adaptive optical system with a liquid mirror.

A deformable mirror based on internal reflection from an electrostatically deformable liquid-air interface is proposed and investigated. A differential equation describing the static behavior of such a mirror is analyzed and solved numerically. Stable closed-loop operation of an adaptive optical system with a liquid deformable mirror is demonstrated, including forming and the correction of low-order aberrations described by Zernike polynomials and the real-time correction of dynamically changing aberrations.

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.

Optimal implementation of a microspectrometer based on a single flat diffraction grating.

An analytical model has been developed and applied to explore the limits in the design of a highly miniaturized planar optical microspectrometer based on an imaging diffraction grating. This design tool has been validated as providing the smallest possible dimensions while maintaining acceptable spectral resolution. The resulting planar spectrometer is composed of two parallel glass plates, which contain all components of the device, including a reflective slit and an imaging diffraction grating. Fabrication is based on microelectromechanical system technology and starts with a single glass wafer; IC-compatible deposition and lithography are applied to realize the parts in aluminum, which makes the microspectrometer highly tolerant for component mismatch. The fabricated spectrometer was mounted directly on top of an image sensor and takes up a volume of only 50 mm(3). The measured spectral resolution of 6 nm (FWHM) in the 100 nm operating wavelength range (600-700 nm) is in agreement with a model calculation.