Application of GRIN-lens-based in-line digital holographic microscopy to automatic detection and localization of particles released from a MEMS device.

Recently, we developed a compact and easy-to-implement in-line digital holographic microscope (DHM) using a GRIN rod lens, which provides better resolution (1.3 µm) compared with commonly used pinhole-based DHM setups. Here, we employ this microscope to acquire 3D holographically reconstructed images of silica microparticles, within the 10-300 µm size range, launched/released from a microelectro-mechanical systems (MEMS) device. To the best of our knowledge, this is the first time that a MEMS device is implemented to store and launch microparticles. The custom-designed MEMS device consists of a 50 µm thick flat circular silicon ultrasonic membrane mounted on an off-the-shelf piezoelectric transducer. Moreover, we propose and experimentally demonstrate a new automatic hybrid detection and localization method for particle field holography that benefits from a combination of well-known minimum intensity and variance of gray level distribution focus metrics. This robust method is fast and provides precise d e p t h/z position of particles. The proposed method is applied for particle testing of the MEMS device, reconstructing 3D visualization, and measuring the size and velocity of released particles. The obtained experimental results show that the velocity of released particles, previously dry-loaded onto the MEMS device, is of the order of a few tens of cm/s.

GRIN-lens-based in-line digital holographic microscopy.

In-line digital holographic microscopy (DHM) provides three-dimensional images with large fields of view and depths of field and micrometer-scale resolution, using a compact, cost-effective, and stable setup. Here, we develop the theoretical background and experimentally demonstrate an in-line DHM based on a gradient-index (GRIN) rod lens. In addition, we develop a conventional pinhole-based in-line DHM with different configurations to compare the resolution and image quality of both GRIN-based and pinhole-based systems. We show that in a high-magnification regime, where the sample is positioned near a source that produces spherical waves, our optimized GRIN-based setup provides better resolution (∼1.38µm). Furthermore, we employed this microscope to holographically image dilute polystyrene micro-particles with diameters of 3.0 and 2.0 µm. We investigated the effect of light source-detector and sample-detector distances on the resolution, by both theory and experiment. Our theoretical and experimental results are in good agreement.

Locally adaptive super-resolution through spatially variant interpolation.

Systems that do not meet the requirements of the sampling theorem produce images corrupted by aliasing. Higher resolution images are attainable by unfolding aliased spatial frequencies. Multiple-image super-resolution has seen much attention in the literature though with no clear optimum algorithm for many real-world applications. We propose a method of multiframe super-resolution using a set of convolutional sinc kernels, tailored to the specific shifts between images, capable of resolving up to the diffraction limit. We demonstrate our method for the case of global shifts before we treat a pixel-level super-resolution.

Development of a prototype active optics system for future space telescopes.

It is envisaged that future large space telescopes will be lightweight and employ active optics to maintain optical quality throughout the mission lifetime. We have proposed a 4 m, two-mirror space telescope with an active optics system based on reimaging the telescope primary mirror onto a small active mirror (110 mm optical pupil). Using Zemax, we demonstrate the feasibility of using this mirror to correct low-order Zernike aberrations and show that the aberration is well corrected across the 2.5 arcmin field of the telescope, operating at 0.55 µm. We describe the modeling carried out to develop the active mirror design. Using end-to-end modeling, a 25-actuator mirror with polar actuator geometry, and a ratio of mechanical to optical pupil diameter of 2 has been chosen. A single-actuator prototype has been manufactured and used to test stroke, linearity, and hysteresis. Finally, we describe the design of a laboratory breadboard that will image phase screens onto an exact replica of the space active mirror and show the results of measuring the phase screen accuracy.

Estimating axial resolution with diffraction theory.

Depth estimation is a classic machine vision and image processing problem aiming at mapping the distances of objects from the camera. The accuracy of this depth map depends on the axial resolution achieved by the system, which is usually estimated using geometrical optics theory. This paper proposes a novel formula using diffraction theory. A comparison with the geometrical approach for estimating the axial resolution is provided, and results for several simulations are discussed.

Registration for images in the presence of additive and multiplicative fixed-pattern noise.

Image registration under conditions of fixed-pattern noise is a difficult problem that has not been solved in the literature. While traditional registration methods are adequate for additive random noise, these are not suited to spatially invariant noise that is additive or multiplicative. We present a method for image registration using a difference operation in the frequency domain. Shift values are then computed by dividing by the object Fourier transform and inverse transforming. The method described is valid for both additive and multiplicative noise and determines shifts with sub-pixel accuracy. Additionally, minimal prior knowledge of the corrupting pattern is required. We compare our method with previous registration methods for varying amounts of noise. Results are presented for both simulated images and images recorded from a thermal camera with significant fixed-pattern noise.

Analysis of Cone Mosaic Reflectance Properties in Healthy Eyes and in Eyes With Nonproliferative Diabetic Retinopathy Over Time.

Purpose: We investigate the reflectance properties of the cone mosaic in adaptive optics (AO) images of healthy subjects and subjects with nonproliferative diabetic retinopathy (NPDR) over time. Methods: We acquired images of the parafoveal cone mosaic over 5 years in 12 healthy subjects and in six patients with mild NPDR. We analyzed the parameters of the cone intensity histogram distribution (mean, SD, and skewness), two metrics of the cone mosaic texture (sharpness and entropy), and two novel metrics (cone/intercone intensity and slope of the variogram). Each metric was calculated on the same four retinal locations (200 × 200 µm areas, 2° from the fovea along the four meridians) over time for each subject. Results: The histogram distributions of cone intensities were similar between the two study groups. However, the cone/intercone intensity, slope of the variograms and entropy showed a significant difference between healthy and NPDR subjects (P = 0.036, P = 0.002, P = 0.014, respectively). All parameters, except for mean cone intensity, did not change with time in this study. Conclusions: We observed significant differences in cone mosaic reflectance properties between healthy eyes and eyes with NPDR, in its spatial organization and in its intensity, especially between directional and nondirectional backscattering. We introduced a novel method for the study of the spatial distribution of cone reflectance, the variogram, which was able to quantify differences of the spatial dependence of cone intensities over a short range between NPDR and healthy eyes.

Phytochrome A and B Regulate Primary Metabolism in Arabidopsis Leaves in Response to Light.

Primary metabolism is closely linked to plant productivity and quality. Thus, a better understanding of the regulation of primary metabolism by photoreceptors has profound implications for agricultural practices and management. This study aims at identifying the role of light signaling in the regulation of primary metabolism, with an emphasis on starch. We first screened seven cryptochromes and phytochromes mutants for starch phenotype. The phyAB mutant showed impairment in starch accumulation while its biomass, chlorophyll fluorescence parameters, and leaf anatomy were unaffected, this deficiency being present over the whole vegetative growth period. Mutation of plastidial nucleoside diphosphate kinase-2 (NDPK2), acting downstream of phytochromes, also caused a deficit in starch accumulation. Besides, the glucose-1-phosphate adenylyltransferase small subunit (APS1) was down-regulated in phyAB. Those results suggest that PHYAB affect starch accumulation through NDPK2 and APS1. Then, we determined changes in starch and primary metabolites in single phyA, single phyB, double phyAB grown in light conditions differing in light intensity and/or light spectral content. PHYA is involved in starch accumulation in all the examined light conditions, whereas PHYB only exhibits a role under low light intensity (44 ± 1 µmol m-2 s-1) or low R:FR (11.8 ± 0.6). PCA analysis of the metabolic profiles in the mutants and wild type (WT) suggested that PHYB acts as a major regulator of the leaf metabolic status in response to light intensity. Overall, we propose that PHYA and PHYB signaling play essential roles in the control of primary metabolism in Arabidopsis leaves in response to light.

Reliability and Agreement Between Metrics of Cone Spacing in Adaptive Optics Images of the Human Retinal Photoreceptor Mosaic.

Purpose: To assess reliability and agreement among three metrics used to evaluate the distribution of cell distances in adaptive optics (AO) images of the cone mosaic. Methods: Using an AO flood illumination retinal camera, we acquired images of the cone mosaic in 20 healthy subjects and 12 patients with retinal diseases. The three spacing metrics studied were the center-to-center spacing (Scc), the local cone spacing (LCS), and the density recovery profile distance (DRPD). Each metric was calculated in sampling areas of different sizes (64 × 64 µm and 204 × 204 µm) across the parafovea. Results: Both Scc and LCS were able to discriminate between healthy subjects and patients with retinal diseases; DRPD did not reliably detect any abnormality in the distribution of cell distances in patients with retinal diseases. The agreement between Scc and LCS was high in healthy subjects (intraclass correlation coefficient [ICC] ≥ 0.79) and moderate in patients with retinal diseases (ICC ≤ 0.51). The DRPD had poor agreement with Scc (ICC ≤ 0.47) and LCS (ICC ≤ 0.37). The correlation between the spacing metrics of the two sampling areas was greater in healthy subjects than in patients with retinal diseases. Conclusions: The Scc and LCS provided interchangeable estimates of cone distance in AO retinal images of healthy subjects but could not be used interchangeably when investigating retinal diseases with significant cell reflectivity loss (≥30%). The DRPD was unreliable for describing cell distance in a human retinal cone mosaic and did not correlate with Scc and LCS. Caution is needed when comparing spacing metrics evaluated in sampling areas of different sizes.

Understanding the changes of cone reflectance in adaptive optics flood illumination retinal images over three years.

Although there is increasing interest in the investigation of cone reflectance variability, little is understood about its characteristics over long time scales. Cone detection and its automation is now becoming a fundamental step in the assessment and monitoring of the health of the retina and in the understanding of the photoreceptor physiology. In this work we provide an insight into the cone reflectance variability over time scales ranging from minutes to three years on the same eye, and for large areas of the retina (≥ 2.0 × 2.0 degrees) at two different retinal eccentricities using a commercial adaptive optics (AO) flood illumination retinal camera. We observed that the difference in reflectance observed in the cones increases with the time separation between the data acquisitions and this may have a negative impact on algorithms attempting to track cones over time. In addition, we determined that displacements of the light source within 0.35 mm of the pupil center, which is the farthest location from the pupil center used by operators of the AO camera to acquire high-quality images of the cone mosaic in clinical studies, does not significantly affect the cone detection and density estimation.

Performance analysis of cone detection algorithms.

Many algorithms have been proposed to help clinicians evaluate cone density and spacing, as these may be related to the onset of retinal diseases. However, there has been no rigorous comparison of the performance of these algorithms. In addition, the performance of such algorithms is typically determined by comparison with human observers. Here we propose a technique to simulate realistic images of the cone mosaic. We use the simulated images to test the performance of three popular cone detection algorithms, and we introduce an algorithm which is used by astronomers to detect stars in astronomical images. We use Free Response Operating Characteristic (FROC) curves to evaluate and compare the performance of the four algorithms. This allows us to optimize the performance of each algorithm. We observe that performance is significantly enhanced by up-sampling the images. We investigate the effect of noise and image quality on cone mosaic parameters estimated using the different algorithms, finding that the estimated regularity is the most sensitive parameter.

Registration of adaptive optics corrected retinal nerve fiber layer (RNFL) images.

Glaucoma is the leading cause of preventable blindness in the western world. Investigation of high-resolution retinal nerve fiber layer (RNFL) images in patients may lead to new indicators of its onset. Adaptive optics (AO) can provide diffraction-limited images of the retina, providing new opportunities for earlier detection of neuroretinal pathologies. However, precise processing is required to correct for three effects in sequences of AO-assisted, flood-illumination images: uneven illumination, residual image motion and image rotation. This processing can be challenging for images of the RNFL due to their low contrast and lack of clearly noticeable features. Here we develop specific processing techniques and show that their application leads to improved image quality on the nerve fiber bundles. This in turn improves the reliability of measures of fiber texture such as the correlation of Gray-Level Co-occurrence Matrix (GLCM).

Pre-processing, registration and selection of adaptive optics corrected retinal images.

PURPOSE: In this paper, the aim is to demonstrate enhanced processing of sequences of fundus images obtained using a commercial AO flood illumination system. The purpose of the work is to (1) correct for uneven illumination at the retina (2) automatically select the best quality images and (3) precisely register the best images. METHODS: Adaptive optics corrected retinal images are pre-processed to correct uneven illumination using different methods; subtracting or dividing by the average filtered image, homomorphic filtering and a wavelet based approach. These images are evaluated to measure the image quality using various parameters, including sharpness, variance, power spectrum kurtosis and contrast. We have carried out the registration in two stages; a coarse stage using cross-correlation followed by fine registration using two approaches; parabolic interpolation on the peak of the cross-correlation and maximum-likelihood estimation. The angle of rotation of the images is measured using a combination of peak tracking and Procrustes transformation. RESULTS: We have found that a wavelet approach (Daubechies 4 wavelet at 6th level decomposition) provides good illumination correction with clear improvement in image sharpness and contrast. The assessment of image quality using a 'Designer metric' works well when compared to visual evaluation, although it is highly correlated with other metrics. In image registration, sub-pixel translation measured using parabolic interpolation on the peak of the cross-correlation function and maximum-likelihood estimation are found to give very similar results (RMS difference 0.047 pixels). We have confirmed that correcting rotation of the images provides a significant improvement, especially at the edges of the image. We observed that selecting the better quality frames (e.g. best 75% images) for image registration gives improved resolution, at the expense of poorer signal-to-noise. The sharpness map of the registered and de-rotated images shows increased sharpness over most of the field of view. CONCLUSION: Adaptive optics assisted images of the cone photoreceptors can be better pre-processed using a wavelet approach. These images can be assessed for image quality using a 'Designer Metric'. Two-stage image registration including correcting for rotation significantly improves the final image contrast and sharpness.

Adaptive optics technology for high-resolution retinal imaging.

Adaptive optics (AO) is a technology used to improve the performance of optical systems by reducing the effects of optical aberrations. The direct visualization of the photoreceptor cells, capillaries and nerve fiber bundles represents the major benefit of adding AO to retinal imaging. Adaptive optics is opening a new frontier for clinical research in ophthalmology, providing new information on the early pathological changes of the retinal microstructures in various retinal diseases. We have reviewed AO technology for retinal imaging, providing information on the core components of an AO retinal camera. The most commonly used wavefront sensing and correcting elements are discussed. Furthermore, we discuss current applications of AO imaging to a population of healthy adults and to the most frequent causes of blindness, including diabetic retinopathy, age-related macular degeneration and glaucoma. We conclude our work with a discussion on future clinical prospects for AO retinal imaging.

On Advanced Estimation Techniques for Exoplanet Detection and Characterization Using Ground-based Coronagraphs.

The direct imaging of planets around nearby stars is exceedingly difficult. Only about 14 exoplanets have been imaged to date that have masses less than 13 times that of Jupiter. The next generation of planet-finding coronagraphs, including VLT-SPHERE, the Gemini Planet Imager, Palomar P1640, and Subaru HiCIAO have predicted contrast performance of roughly a thousand times less than would be needed to detect Earth-like planets. In this paper we review the state of the art in exoplanet imaging, most notably the method of Locally Optimized Combination of Images (LOCI), and we investigate the potential of improving the detectability of faint exoplanets through the use of advanced statistical methods based on the concepts of the ideal observer and the Hotelling observer. We propose a formal comparison of techniques using a blind data challenge with an evaluation of performance using the Receiver Operating Characteristic (ROC) and Localization ROC (LROC) curves. We place particular emphasis on the understanding and modeling of realistic sources of measurement noise in ground-based AO-corrected coronagraphs. The work reported in this paper is the result of interactions between the co-authors during a week-long workshop on exoplanet imaging that was held in Squaw Valley, California, in March of 2012.

Enhanced faint companion photometry and astrometry using wavelength diversity.

In this paper we examine approaches to faint companion detection and estimation in multi-spectral images. We will employ the Hotelling observer, which is the optimal linear algorithm for signal detection. We have shown how to use this observer to estimate faint object position and brightness in the presence of residual speckle, which usually limits astrometric and photometric techniques. These speckles can be reduced by differential imaging techniques such as Angular Differential Imaging and Spectral Differential Imaging. Here we present results based on simulations of adaptive-optics-corrected images from an Extremely Large Telescope (ELT) that contain quasi-static speckle noise. The simulation includes Angular Differential Imaging and Spectral Differential Imaging to reduce the residual speckle and subsequent multi-wavelength processing. We examine the feasibility of this approach on simulated ELT observations of faint companions.

Experimental detection of optical vortices with a Shack-Hartmann wavefront sensor.

Laboratory experiments are carried out to detect optical vortices in conditions typical of those experienced when a laser beam is propagated through the atmosphere. A Spatial Light Modulator (SLM) is used to mimic atmospheric turbulence and a Shack-Hartmann wavefront sensor is utilised to measure the slopes of the wavefront surface. A matched filter algorithm determines the positions of the Shack-Hartmann spot centroids more robustly than a centroiding algorithm. The slope discrepancy is then obtained by taking the slopes measured by the wavefront sensor away from the slopes calculated from a least squares reconstruction of the phase. The slope discrepancy field is used as an input to the branch point potential method to find if a vortex is present, and if so to give its position and sign. The use of the slope discrepancy technique greatly improves the detection rate of the branch point potential method. This work shows the first time the branch point potential method has been used to detect optical vortices in an experimental setup.

Correction of ocular and atmospheric wavefronts: a comparison of the performance of various deformable mirrors.

The main applications of adaptive optics are the correction of the effects of atmospheric turbulence on ground-based telescopes and the correction of ocular aberrations in retinal imaging and visual simulation. The requirements for the wavefront corrector, usually a deformable mirror, will depend on the statistics of the aberrations to be corrected; here we compare the spatial statistics of wavefront aberrations expected in these two applications. We also use measured influence functions and numerical simulations to compare the performance of eight commercially available deformable mirrors for these tasks. The performance is studied as a function of the size of the optical pupil relative to the actuated area of the mirrors and as a function of the number of modes corrected. In the ocular case it is found that, with the exception of segmented mirrors, the performance is greatly enhanced by having a ring of actuators outside the optical pupil, as this improves the correction of the pupil edge. The effect is much smaller in the case of Kolmogorov wavefronts. It is also found that a high Strehl ratio can be obtained in the ocular case with a relatively low number of actuators if the stroke is sufficient. Increasing the number of actuators has more importance in the Kolmogorov case, even for the relatively weak turbulence considered here.

Chromatic effects of the atmosphere on astronomical adaptive optics.

The atmosphere introduces chromatic errors that may limit the performance of adaptive optics (AO) systems on large telescopes. Various aspects of this problem have been considered in the literature over the past two decades. It is necessary to revisit this problem in order to examine the effect on currently planned systems, including very high-order AO on current 8-10 m class telescopes and on future 30-42 m extremely large telescopes. We review the literature on chromatic effects and combine an analysis of all effects in one place. We examine implications for AO and point out some effects that should be taken into account in the design of future systems. In particular we show that attention should be paid to chromatic pupil shifts, which may arise in components such as atmospheric dispersion compensators.

Application of the Hotelling and ideal observers to detection and localization of exoplanets.

The ideal linear discriminant or Hotelling observer is widely used for detection tasks and image-quality assessment in medical imaging, but it has had little application in other imaging fields. We apply it to detection of planets outside of our solar system with long-exposure images obtained from ground-based or space-based telescopes. The statistical limitations in this problem include Poisson noise arising mainly from the host star, electronic noise in the image detector, randomness or uncertainty in the point-spread function (PSF) of the telescope, and possibly a random background. PSF randomness is reduced but not eliminated by the use of adaptive optics. We concentrate here on the effects of Poisson and electronic noise, but we also show how to extend the calculation to include a random PSF. For the case where the PSF is known exactly, we compare the Hotelling observer to other observers commonly used for planet detection; comparison is based on receiver operating characteristic (ROC) and localization ROC (LROC) curves.