Showing papers on "Point spread function published in 2014"

Isotropic three-dimensional super-resolution imaging with a self-bending point spread function

Abstract: Airy beams maintain their intensity profiles over a large propagation distance without substantial diffraction and exhibit lateral bending during propagation1-5. This unique property has been exploited for micromanipulation of particles6, generation of plasma channels7 and guidance of plasmonic waves8, but has not been explored for high-resolution optical microscopy. Here, we introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional (3D) super-resolution fluorescence imaging. We designed a side-lobe-free SB-PSF and implemented a two-channel detection scheme to enable unambiguous 3D localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF well suited for precise 3D localization of molecules over a large imaging depth. Using this method, we obtained super-resolution imaging with isotropic 3D localization precision of 10-15 nm over a 3 μm imaging depth from ∼2000 photons per localization.

Embedded pupil function recovery for Fourier ptychographic microscopy

Abstract: We develop and test a pupil function determination algorithm, termed embedded pupil function recovery (EPRY), which can be incorporated into the Fourier ptychographic microscopy (FPM) algorithm and recover both the Fourier spectrum of sample and the pupil function of imaging system simultaneously. This EPRY-FPM algorithm eliminates the requirement of the previous FPM algorithm for a priori knowledge of the aberration in the imaging system to reconstruct a high quality image. We experimentally demonstrate the effectiveness of this algorithm by reconstructing high resolution, large field-of-view images of biological samples. We also illustrate that the pupil function we retrieve can be used to study the spatially varying aberration of a large field-of-view imaging system. We believe that this algorithm adds more flexibility to FPM and can be a powerful tool for the characterization of an imaging system’s aberration.

Optimal point spread function design for 3D imaging.

Abstract: Optical imaging of single nanoscale objects such as a quantum dot, metallic nanoparticle, or a single molecule provides a powerful window into a variety of biological or material systems, and the physical problem of extracting maximum information from single emitters is an important goal. One application is single-particle tracking (SPT [1]), which relies upon extracting the spatial trajectory of a single moving molecular label, quantum dot, or metallic nanoparticle from a series of images. For example, a single mRNA particle can be localized and followed in a living cell in real-time [2]. Another application of single-molecule localization is “super-resolution” (SR) microscopy, [3–5] which works by ensuring that only a sparse subset of labels on an extended object (e.g. a cellular structure) are emitting in each imaging frame. One localizes the single emitters just as in SPT; the multitude of localizations are then reconstructed into a single, high-resolution image. This enables the spatial resolving power of SR microscopy to surpass the classical diffraction resolution limit by 5- to 10-fold. Historically, single-particle localization was used for 2D imaging, namely, inferring the x,y coordinates of each emitter, e.g. by centroid-fitting of by fitting to a 2D Gaussian [6]. However, the third spatial dimension, z, or the depth of an emitter, can also be inferred from its measured 2D image. This can be done by considering how the shape of the microscope’s point spread function (PSF) varies with emitter position. The PSF of a microscope is the image that is detected when observing a point source. For a standard microscope, to a good approximation, the PSF in focus (i.e. z=0) resembles a circular Airy pattern, and its shape is invariant to lateral shifts (x,y) of the emitter – however it will change upon defocus (z). Unfortunately, the standard PSF spreads out (defocuses) quickly with z which limits the range over which z can be determined. Importantly, to obtain much more useful 3D position information, the PSF of the microscope can be altered – for example by pupil (Fourier) plane processing [7,8]. Phase modulating the electromagnetic field in the Fourier plane is a low-loss method to encode z-information in the shape of the image on the camera. Examples of this include astigmatic PSFs [9,10], double-helix (DH-PSF) microscopy [11–13] or segmented phase ramps [14]. The precision to which a single emitter can be localized depends on several factors. These include the emitter’s brightness (detected photon flux), background fluorescence, detector pixel size, and detection noise [15,16]. Another key factor is the shape of the PSF itself. For example, in astigmatism-based 3D imaging, the PSF is altered to have an elliptical shape, and the z position of the emitter can be determined by the relative widths of the PSF along the two principal axes [9,10]. The double-helix PSF [12,13] is composed of two spots, with the angle between a line connecting them and the camera axis encoding the z position of the emitter. Among existing PSFs for 3D imaging, the double-helix PSF has been shown to allow a larger depth of field than astigmatism (~2–3 μm vs ~0.5–0.7 μm) [17], and a recently suggested PSF based on accelerating beams [18] demonstrates high, uniform precision over a 3 μm range. The purpose of this paper is to fundamentally improve upon these previous schemes. Here, we address the problem of finding a feasible and optimally informative PSF. Namely, we ask the question – given an imaging scenario with certain characteristics (e.g. magnification, noise level, pixel size, emitter signal) – what is the pupil plane pattern that would yield maximal physical information about the 3D position of an emitter, and what is the resulting optimal PSF? In other words – since localization precision depends on the PSF of the system – can we design the system to have a PSF that would yield the best possible precision in determining x, y, and z, compared to any other PSF? We regard such a PSF as optimally informative. A powerful measure of the effectiveness of a PSF for encoding an emitter’s position is based on Fisher information [17,19,20], a concept from statistical information theory. Fisher information is a mathematical measure of the sensitivity of an observable quantity (the PSF) to changes in its underlying parameters (emitter position). Using the Fisher information function, one may compute the Cramer-Rao lower bound (CRLB), which is the theoretical best-case x,y,z precision that can be attained (with any unbiased estimator) given a PSF and a noise model. With the right estimator, the best-case localization precision represented by the CRLB can be approached in practice [21–23]. Traditionally, the CRLB has been used as an analysis tool, i.e. to evaluate the performance of an existing PSF design, which is often conceived using physical intuition and reasonable requirements (e.g. a significant change of the PSF over the z-range of interest, and concentration of emitted light into small spots). The CRLB has also been used to fine-tune an existing PSF [24]. To find the optimal pupil plane pattern, and thereby the optimal PSF, we propose a new approach to PSF design –we treat the PSF as a free design parameter of the imaging system, and generate PSFs with optimal photon-efficient 3D position encoding, with no prior constraints on the shape of the PSF. This is achieved by CRLB optimization – that is, we directly solve the mathematical optimization problem of minimizing the CRLB (and hence improving the precision bound) of the system, and use the resulting PSF. Such a PSF will provide optimal precision by definition. Physically reasonable requirements are accounted for by using realistic imaging and noise models, including pixelation, photon shot-noise Poisson statistics, and background fluorescence. This enables us to demonstrate, for typical experimental conditions and without scanning, the highest theoretical precision to date over a 3 μm axial range, as well as <50 nm experimental precision for an unprecedented ~5 μm range.

Aperture-scanning Fourier ptychography for 3D refocusing and super-resolution macroscopic imaging

Abstract: We report an imaging scheme, termed aperture-scanning Fourier ptychography, for 3D refocusing and super-resolution macroscopic imaging The reported scheme scans an aperture at the Fourier plane of an optical system and acquires the corresponding intensity images of the object The acquired images are then synthesized in the frequency domain to recover a high-resolution complex sample wavefront; no phase information is needed in the recovery process We demonstrate two applications of the reported scheme In the first example, we use an aperture-scanning Fourier ptychography platform to recover the complex hologram of extended objects The recovered hologram is then digitally propagated into different planes along the optical axis to examine the 3D structure of the object We also demonstrate a reconstruction resolution better than the detector pixel limit (ie, pixel super-resolution) In the second example, we develop a camera-scanning Fourier ptychography platform for super-resolution macroscopic imaging By simply scanning the camera over different positions, we bypass the diffraction limit of the photographic lens and recover a super-resolution image of an object placed at the far field This platform’s maximum achievable resolution is ultimately determined by the camera’s traveling range, not the aperture size of the lens The FP scheme reported in this work may find applications in 3D object tracking, synthetic aperture imaging, remote sensing, and optical/electron/X-ray microscopy

UVMULTIFIT: A versatile tool for fitting astronomical radio interferometric data

Abstract: Context. The analysis of astronomical interferometric data is often performed on the images obtained after deconvolving the interferometer’s point spread function. This strategy can be understood (especially for cases of sparse arrays) as fitting models to models, since the deconvolved images are already non-unique model representations of the actual data (i.e., the visibilities). Indeed, the interferometric images may be affected by visibility gridding, weighting schemes (e.g., natural vs. uniform), and the particulars of the (non-linear) deconvolution algorithms. Fitting models to the direct interferometric observables (i.e., the visibilities) is preferable in the cases of simple (analytical) sky intensity distributions. Aims. We present UVMULTIFIT, a versatile library for fitting visibility data, implemented in a Python-based framework. Our software is currently based on the CASA package, but can be easily adapted to other analysis packages, provided they have a Python API. Methods. The user can simultaneously fit an indefinite number of source components to the data, each of which depend on any algebraic combination of fitting parameters. Fits to individual spectral-line channels or simultaneous fits to all frequency channels are allowed. Results. We have tested the software with synthetic data and with real observations. In some cases (e.g., sources with sizes smaller than the diffraction limit of the interferometer), the results from the fit to the visibilities (e.g., spectra of close by sources) are far superior to the output obtained from the mere analysis of the deconvolved images. Conclusions. UVMULTIFIT is a powerful improvement of existing tasks to extract the maximum amount of information from visibility data, especially in cases close to the sensitivity/resolution limits of interferometric observations.

High-resolution fluorescence imaging via pattern-illuminated Fourier ptychography

Abstract: Fluorescence microscopy plays a vital role in modern biological research and clinical diagnosis. Here, we report an imaging approach, termed pattern-illuminated Fourier ptychography (FP), for fluorescence imaging beyond the diffraction limit of the employed optics. This approach iteratively recovers a high-resolution fluorescence image from many pattern-illuminated low-resolution intensity measurements. The recovery process starts with one low-resolution measurement as the initial guess. This initial guess is then sequentially updated by other measurements, both in the spatial and Fourier domains. In the spatial domain, we use the pattern-illuminated low-resolution images as intensity constraints for the sample estimate. In the Fourier domain, we use the incoherent optical-transfer-function of the objective lens as the object support constraint for the solution. The sequential updating process is then repeated until the sample estimate converges, typically for 5-20 times. Different from the conventional structured illumination microscopy, any unknown pattern can be used for sample illumination in the reported framework. In particular, we are able to recover both the high-resolution sample image and the unknown illumination pattern at the same time. As a demonstration, we improved the resolution of a conventional fluorescence microscope beyond the diffraction limit of the employed optics. The reported approach may provide an alternative solution for structure illumination microscopy and find applications in wide-field, high-resolution fluorescence imaging.

Extending Single-Molecule Microscopy Using Optical Fourier Processing

Abstract: This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules.

The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging.

Abstract: Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

High-throughput ptychography using Eiger: scanning X-ray nano-imaging of extended regions

Abstract: The smaller pixel size and high frame rate of next-generation photon counting pixel detectors opens new opportunities for the application of X-ray coherent diffractive imaging (CDI). In this manuscript we demonstrate fast image acquisition for ptychography using an Eiger detector. We achieve above 25,000 resolution elements per second, or an effective dwell time of 40 μs per resolution element, when imaging a 500 μm × 290 μm region of an integrated electronic circuit with 41 nm resolution. We further present the application of a scheme of sharing information between image parts that allows the field of view to exceed the range of the piezoelectric scanning system and requirements on the stability of the illumination to be relaxed.

Ultra Low Surface Brightness Imaging with the Dragonfly Telephoto Array

Abstract: We describe the Dragonfly Telephoto Array, a robotic imaging system optimized for the detection of extended ultra low surface brightness structures. The array consists of eight Canon 400mm f/2.8 telephoto lenses coupled to eight science-grade commercial CCD cameras. The lenses are mounted on a common framework and are co-aligned to image simultaneously the same position on the sky. The system provides an imaging capability equivalent to a 0.4m aperture f/1.0 refractor with a 2.6 deg X 1.9 deg field of view. The system has no obstructions in the light path, optimized baffling, and internal optical surfaces coated with a new generation of anti-reflection coatings based on sub-wavelength nanostructures. As a result, the array's point spread function has a factor of ~10 less scattered light at large radii than well-baffled reflecting telescopes. The Dragonfly Telephoto Array is capable of imaging extended structures to surface brightness levels below 30 mag/arcsec^2 in 10h integrations (without binning or foreground star removal). This is considerably deeper than the surface brightness limit of any existing wide-field telescope. At present no systematic errors limiting the usefulness of much longer integration times has been identified. With longer integrations (50-100h), foreground star removal and modest binning the Dragonfly Telephoto Array is capable of probing structures with surface brightnesses below 32 mag/arcsec^2. Detection of structures at these surface brightness levels may hold the key to solving the "missing substructure" and "missing satellite" problems of conventional hierarchical galaxy formation models. The Dragonfly Telephoto Array is therefore executing a fully-automated multi-year imaging survey of a complete sample of nearby galaxies in order to undertake the first census of ultra-faint substructures in the nearby Universe.

Harnessing the Point-Spread Function for High-Resolution Far-Field Optical Microscopy

Abstract: The resolution limit of far-field optical microscopy is reexamined with a full vectorial theoretical analysis. A highly symmetric excitation optical field and optimized detection scheme are proposed to harness the total point-spread function for a microscopic system. Spatial resolution of better than $1/6\ensuremath{\lambda}$ is shown to be obtainable, giving rise to a resolution better than 100 nm with visible light excitation. The experimental measurement is applied to examine nonfluorescent samples. A lateral resolution of $1/5\ensuremath{\lambda}$ is obtained in truly far-field optical microscopy with a working distance greater than $\ensuremath{\sim}500\ensuremath{\lambda}$. Comparison is made for the far-field microscopic measurement with that of a nearfield scanning optical microscopy, showing that the proposed scheme provides a better image quality.

The influence of diffuse scattered light I. The PSF and its role in observations of the edge-on galaxy NGC 5907

Abstract: All telescopes and instruments are to some degree affected by scattered light. It is possible to estimate the amount of such scattered light, and even correct for it, with a radially extended point spread function (PSF). The outer parts of the PSF have only rarely been determined, since they are faint and therefore difficult to measure. A mostly complete overview of existing properties and measurements of radially extended PSFs is presented, to both show their similarities and to indicate how bright extended objects can be used to measure the faintest regions. The importance of the far wings of the PSF and their possible temporal variations are demonstrated in three edge-on galaxy models. The same study is applied to the first edge-on galaxy where earlier observations reveal a halo, NGC 5907. All PSFs were collected in two diagrams, after they were offset or normalized, when that was possible. Surfacebrightness structures of edge-on galaxies were modelled and analysed to study scattered-light haloes that result when there is an exponential disc. The models were convolved with both a lower-limit PSF and a more average PSF. The PSF of the observed data could be used in the case of NGC 5907. The comparison of the PSFs demonstrates a lower-limit r −2 power-law decline at larger radii. The analysis of the galaxy models shows that the outer parts of the PSF also are important to correctly model and analyse observations and, in particular, fainter regions. The reassessed analysis of the earlier measurements of NGC 5907 reveals an explanation for the faint halo in scattered light, within the quoted level of accuracy.

Method and apparatus for scanning with controlled spherical aberration

Abstract: A reader obtains image data corresponding to an image of optically encoded information that is received via a lens unit that causes controlled spherical aberration blurring that is precisely known. The reader may perform deconvolution processing on the image data to render it decodable. The deconvolution processing may implement a Weiner filter that uses data corresponding to a near-field point spread function of the lens unit. The depth of field of the reader is greater than that of conventional reader in all lighting conditions.

High-speed terahertz imaging toward food quality inspection

Abstract: In contrast to conventional x-ray food inspection systems that have difficulty in detecting low-density materials, a terahertz imaging system can even identify insects and plastics embedded in a food matrix. A reflection-mode continuous-wave terahertz imaging system was therefore developed for application to food quality inspection, which requires fast, compact, and low-cost detection. High-speed operation of the terahertz imaging system was achieved through the use of a beam-steering tool. A reasonable compromise between the spatial resolution and the scan length of an aspheric f-theta scanning lens could be achieved by optimizing the lens parameters.

UVMULTIFIT: A versatile tool for fitting astronomical radio interferometric data

Abstract: The analysis of astronomical interferometric data is often performed on the images obtained after deconvolution of the interferometer's point spread function (PSF). This strategy can be understood (especially for cases of sparse arrays) as fitting models to models, since the deconvolved images are already non-unique model representations of the actual data (i.e., the visibilities). Indeed, the interferometric images may be affected by visibility gridding, weighting schemes (e.g., natural vs. uniform), and the particulars of the (non-linear) deconvolution algorithms. Fitting models to the direct interferometric observables (i.e., the visibilities) is preferable in the cases of simple (analytical) sky intensity distributions. In this paper, we present UVMULTIFIT, a versatile library for fitting visibility data, implemented in a Python-based framework. Our software is currently based on the CASA package, but can be easily adapted to other analysis packages, provided they have a Python API. We have tested the software with synthetic data, as well as with real observations. In some cases (e.g., sources with sizes smaller than the diffraction limit of the interferometer), the results from the fit to the visibilities (e.g., spectra of close by sources) are far superior to the output obtained from the mere analysis of the deconvolved images. UVMULTIFIT is a powerful improvement of existing tasks to extract the maximum amount of information from visibility data, especially in cases close to the sensitivity/resolution limits of interferometric observations.

The influence of diffuse scattered light I. The PSF and its role to observations of the edge-on galaxy NGC 5907

Abstract: All telescopes and instruments are to some degree affected by scattered light. It is possible to estimate the amount of such scattered light, and even correct for it, with a radially extended point spread function (PSF). The outer parts of the PSF have only rarely been determined, since they are faint and therefore difficult to measure. A mostly complete overview of existing properties and measurements of radially extended PSFs is presented, to both show their similarities and to indicate how bright extended objects can be used to measure the faintest regions. The importance of the far wings of the PSF and their possible temporal variations are demonstrated in three edge-on galaxy models. The same study is applied to the first edge-on galaxy where earlier observations reveal a halo, NGC 5907. All PSFs were collected in two diagrams, after they were offset or normalized, when that was possible. Surface-brightness structures of edge-on galaxies were modelled and analysed to study scattered-light haloes that result with an exponential disc. The models were convolved with both a lower-limit PSF and a more average PSF. The PSF of the observed data could be used in the case of NGC 5907. The comparison of the PSFs demonstrates a lower-limit $r^{-2}$ power-law decline at larger radii. The analysis of the galaxy models shows that also the outer parts of the PSF are important to correctly model and analyse observations and, in particular, fainter regions. The reassessed analysis of the earlier measurements of NGC 5907 reveals an explanation for the faint halo in scattered light, within the quoted level of accuracy.

Updated point spread function simulations for JWST with WebbPSF

Abstract: Accurate models of optical performance are an essential tool for astronomers, both for planning scientific observations ahead of time, and for a wide range of data analysis tasks such as point-spread-function (PSF)-fitting photometry and astrometry, deconvolution, and PSF subtraction. For the James Webb Space Telescope, the WebbPSF program provides a PSF simulation tool in a flexible and easy-to-use software package available to the community and implemented in Python. The latest version of WebbPSF adds new support for spectroscopic modes of JWST NIRISS, MIRI, and NIRSpec, including modeling of slit losses and diffractive line spread functions. It also provides additional options for modeling instrument defocus and/or pupil misalignments. The software infrastructure of WebbPSF has received enhancements including improved parallelization, an updated graphical interface, a better configuration system, and improved documentation. We also present several comparisons of WebbPSF simulated PSFs to observed PSFs obtained using JWST's flight science instruments during recent cryovac tests. Excellent agreement to first order is achieved for all imaging modes cross-checked thus far, including tests for NIRCam, FGS, NIRISS, and MIRI. These tests demonstrate that WebbPSF model PSFs have good fidelity to the key properties of JWST's as-built science instruments.

Performance of the VLT Planet Finder SPHERE - I. Photometry and astrometry precision with IRDIS and IFS in laboratory

Abstract: Context. The new planet finder for the Very Large Telescope (VLT), the Spectro-Polarimetric High-contrast Exoplanet REsearch (SPHERE), just had its first light in Paranal. A dedicated instrument for the direct detection of planets, SPHERE, is composed of a polametric camera in visible light, the Zurich IMager POLarimeter (ZIMPOL), and two near-infrared sub-systems: the Infra-Red Dual-beam Imager and Spectrograph (IRDIS), a multi-purpose camera for imaging, polarimetry, and long-slit spectroscopy, and the integral field spectrograph (IFS), an integral field spectrograph. Aims. We present the results obtained from the analysis of data taken during the laboratory integration and validation phase, after the injection of synthetic planets. Since no continuous field rotation could be performed in the laboratory, this analysis presents results obtained using reduction techniques that do not use the angular differential imaging (ADI) technique. Methods. To perform the simulations, we used the instrumental point spread function (PSF) and model spectra of L and T-type objects scaled in contrast with respect to the host star. We evaluated the expected error in astrometry and photometry as a function of the signal to noise of companions, after spectral differential imaging (SDI) reduction for IRDIS and spectral deconvolution (SD) or principal component analysis (PCA) data reductions for IFS. Results. We deduced from our analysis, for example, that β Picb, a 12 Myr old planet of ~10 MJup and semi-major axis of 9-10 AU, would be detected with IRDIS with a photometric error of 0.16 mag and with a relative astrometric position error of 1.1 mas. With IFS, we could retrieve a spectrum with error bars of about 0.15 mag on each channel and astrometric relative position error of 0.6 mas. For a fainter object such as HR 8799d, a 13 MJup planet at a distance of 27 AU, IRDIS could obtain a relative astrometric error of 3 mas.

Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence.

Abstract: The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms.

Neutron source reconstruction from pinhole imaging at National Ignition Facility

Abstract: The neutron imaging system at the National Ignition Facility (NIF) is an important diagnostic tool for measuring the two-dimensional size and shape of the neutrons produced in the burning deuterium-tritium plasma during the ignition stage of inertial confinement fusion (ICF) implosions at NIF. Since the neutron source is small (∼100 μm) and neutrons are deeply penetrating (>3 cm) in all materials, the apertures used to achieve the desired 10-μm resolution are 20-cm long, single-sided tapers in gold. These apertures, which have triangular cross sections, produce distortions in the image, and the extended nature of the pinhole results in a non-stationary or spatially varying point spread function across the pinhole field of view. In this work, we have used iterative Maximum Likelihood techniques to remove the non-stationary distortions introduced by the aperture to reconstruct the underlying neutron source distributions. We present the detailed algorithms used for these reconstructions, the stopping criteria used and reconstructed sources from data collected at NIF with a discussion of the neutron imaging performance in light of other diagnostics.

High resolution surface plasmon resonance imaging for single cells

Abstract: Surface plasmon resonance imaging (SPRI) is a label-free technique that can image refractive index changes at an interface. We have previously used SPRI to study the dynamics of cell-substratum interactions. However, characterization of spatial resolution in 3 dimensions is necessary to quantitatively interpret SPR images. Spatial resolution is complicated by the asymmetric propagation length of surface plasmons in the x and y dimensions leading to image degradation in one direction. Inferring the distance of intracellular organelles and other subcellular features from the interface by SPRI is complicated by uncertainties regarding the detection of the evanescent wave decay into cells. This study provides an experimental basis for characterizing the resolution of an SPR imaging system in the lateral and distal dimensions and demonstrates a novel approach for resolving sub-micrometer cellular structures by SPRI. The SPRI resolution here is distinct in its ability to visualize subcellular structures that are in proximity to a surface, which is comparable with that of total internal reflection fluorescence (TIRF) microscopy but has the advantage of no fluorescent labels. An SPR imaging system was designed that uses a high numerical aperture objective lens to image cells and a digital light projector to pattern the angle of the incident excitation on the sample. Cellular components such as focal adhesions, nucleus, and cellular secretions are visualized. The point spread function of polymeric nanoparticle beads indicates near-diffraction limited spatial resolution. To characterize the z-axis response, we used micrometer scale polymeric beads with a refractive index similar to cells as reference materials to determine the detection limit of the SPR field as a function of distance from the substrate. Multi-wavelength measurements of these microspheres show that it is possible to tailor the effective depth of penetration of the evanescent wave into the cellular environment. We describe how the use of patterned incident light provides SPRI at high spatial resolution, and we characterize a finite limit of detection for penetration depth. We demonstrate the application of a novel technique that allows unprecedented subcellular detail for SPRI, and enables a quantitative interpretation of SPRI for subcellular imaging.

Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

Abstract: In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM.

Wavefront-guided scleral lens correction in keratoconus.

Abstract: Scleral contact lenses were the first form of contact lens correction successfully demonstrated, and Pearson et. al. report almost simultaneous demonstrations by Fick, Kalt and Mueller in the late 1880s.1-3 With the introduction of hydrogel soft contact lenses, gas permeable lens prescription (including scleral contact lenses) reduced as a proportion of the overall contact lens market.4 However, the unique properties of scleral lenses (large diameter, vaulting of the central cornea and the formation of a pre-corneal tear reservoir) have led to renewed specialty application in today'ss clinic.5-12 In particular, clinicians are finding utility in treating abnormal corneal conditions such as pellucid marginal degeneration, keratoconus, dry eye syndrome, post-LASIK ectasia and non-healing epithelial defects.5-12 In the case of the highly aberrated keratoconic eye, practicing clinicians report that scleral contact lenses are finding success due to the combined potential of improved optics, vision and comfort. Rigid gas-permeable lenses reduce higher-order wavefront error associated with keratoconus.13-19 However, during rigid gas-permeable lens wear, the residual higher-order aberrations remain elevated, as compared to normal eyes.13-19 In this feature issue of Optometry and Vision Science, Yang et al. show that for a sample of 20 keratoconic eyes, the correction of residual aberration results in an improvement in contrast sensitivity at low (2 c/d) and intermediate (4, 8 and 16 c/d) spatial frequencies.20 The authors of that work suggest custom contact lenses as a method to achieve this additional reduction in aberration.20 The literature currently contains several demonstrations of customized contact lenses that specifically target higher-order aberration.21-26 For customized higher-order compensating optics to perform optimally, they must be integrated into a stable lens platform and properly aligned to the underlying optical errors. When a lens is not stable or is misaligned, aberration correction is reduced, and can actually lead to an increase in higher-order aberration and a reduction in visual performance.27-29 Work by Sabesan et al. on severe keratoconus subjects demonstrated that higher-order RMS was reduced and both visual acuity and contrast sensitivity improved with wavefront-guided scleral contact lenses (wfgSCLs).26 They also noted that while improved, visual performance did not reach levels seen in individuals that habitually experience normal levels of higher-order aberration. When judging the performance of a wavefront-guided contact lens (regardless of modality), higher-order RMS (HORMS) is a logical metric of optical performance, as these lenses are specifically designed to target higher-order aberrations. In terms of real-world visual performance, HORMS is not an optimal predictor of performance, as it does not consider the contribution of lower-order aberrations (residual uncorrected sphere and cylinder) nor does it consider the interaction between individual aberration terms.30-31 The literature now contains several descriptions of optical quality metrics that examine the interaction of individual terms and the impact on visual performance.32-40 Three metrics: light in the bucket, visual Strehl ratio and neural sharpness, have been shown to be well-correlated with changes in optical performance in keratoconus.40 All three are based on retinal image quality and two of the three incorporate neural weighting functions. Their mathematical formulation (briefly presented here) was previously detailed by Thibos et al.33 The formulation for light in the bucket (LIB) is given in Equation 1. As described by Thibos et al.,33 LIB calculates the percentage of total energy in a normalized point spread function falling in an area defined by the core of a diffraction limited PSF for the same pupil diameter. In essence, it is a measure of compactness of the PSF. LIB=∫DLcorePSFN(x,y)dxdy (1) Visual Strehl ratio computed in the spatial domain (VSX) is given in Equation 2. As described by Thibos et al.,33 VSX is calculated as the inner product of the PSF with a neural weighting function (inverse of contrast sensitivity function), normalized to the diffraction-limited case. VSX attempts to include both optical quality (represented by the PSF) with the efficiency of the visual system at processing the image formed on the retina (represented mathematically by the neural weighting function N). VSX=∫psfPSF(x,y)N(x,y)dxdy∫psfPSFDL(x,y)N(x,y)dxdy (2) Neural sharpness (NS) is given in Equation 3. As described by Thibos et al.,33 this metric was conceptualized by Williams as a way, much like VSX, to capture the effectiveness of a PSF for stimulating the neural portion of the visual system by using a Gaussian weighting function.34 NS=∫psfPSF(x,y)g(x,y)dxdy∫psfPSFDL(x,y)g(x,y)dxdy (3) VSX and NS both neurally weight the PSF, albeit with different weighting functions. As described by Thibos et al.,33 the main difference in the weighting functions being that the weighting function of VSX contains an inhibitory surround outside the PSF core, whereas the weighting function for NS has no such inhibitory surround. The current experiment focuses on the optical quality achieved with scleral contact lenses by reporting metrics of image quality calculated from the combined residual lower-order and higher-order aberrations, using these metrics that are highly correlated with visual acuity in keratoconus.40 Results are compared to similar values for the well-corrected normal population. Importantly, this report also details the clinical process utilized in dispensing wfgSCLs as envisioned by the investigators; this description is included for the interest of the contact lens practitioner that may be interested in fitting these lenses in the future.

An implementation of Bayesian lensing shear measurement

Abstract: The Bayesian gravitational shear estimation algorithm developed by Bernstein and Armstrong (2014) can potentially be used to overcome multiplicative noise bias and recover shear using very low signal-to-noise ratio (S/N) galaxy images. In that work the authors confirmed the method is nearly unbiased in a simplified demonstration, but no test was performed on images with realistic pixel noise. Here I present a full implementation for fitting models to galaxy images, including the effects of a point spread function (PSF) and pixelization. I tested the implementation using simulated galaxy images modeled as Sersic profiles with n=1 (exponential) and n=4 (De Vaucouleurs'), convolved with a PSF and a flat pixel response function. I used a round Gaussian model for the PSF to avoid potential PSF-fitting errors. I simulated galaxies with mean observed, post-PSF full-width at half maximum equal to approximately 1.2 times that of the PSF, with log-normal scatter. I also drew fluxes from a log-normal distribution. I produced independent simulations, each with pixel noise tuned to produce different mean S/N ranging from 10-1000. I applied a constant shear to all images. I fit the simulated images to a model with the true Sersic index to avoid modeling biases. I recovered the input shear with fractional error less than 2 x 10^{-3} in all cases. In these controlled conditions, and in the absence of other multiplicative errors, this implementation is sufficiently unbiased for current surveys and approaches the requirements for planned surveys.

Determining the Resolution Limits of Electron-Beam Lithography: Direct Measurement of the Point-Spread Function

Abstract: One challenge existing since the invention of electron-beam lithography (EBL) is understanding the exposure mechanisms that limit the resolution of EBL. To overcome this challenge, we need to understand the spatial distribution of energy density deposited in the resist, that is, the point-spread function (PSF). During EBL exposure, the processes of electron scattering, phonon, photon, plasmon, and electron emission in the resist are combined, which complicates the analysis of the EBL PSF. Here, we show the measurement of delocalized energy transfer in EBL exposure by using chromatic aberration-corrected energy-filtered transmission electron microscopy (EFTEM) at the sub-10 nm scale. We have defined the role of spot size, electron scattering, secondary electrons, and volume plasmons in the lithographic PSF by performing EFTEM, momentum-resolved electron energy loss spectroscopy (EELS), sub-10 nm EBL, and Monte Carlo simulations. We expect that these results will enable alternative ways to improve the resol...

Breakthroughs in Photonics 2013: Fourier Ptychographic Imaging

Abstract: Fourier ptychography (FP) is a recently developed computational framework for high-resolution high-throughput imaging. In this paper, we will review the latest development of the Fourier ptychographic imaging scheme. We will demonstrate its applications in wide-field imaging, quantitative phase imaging, and adaptive imaging. We will also discuss its potential applications in X-ray optics and transmission electron microscopy.

Optimized approaches for optical sectioning and resolution enhancement in 2D structured illumination microscopy

Abstract: The use of structured illumination in fluorescence microscopy allows the suppression of out of focus light and an increase in effective spatial resolution. In this paper we consider different approaches for reconstructing 2D structured illumination images in order to combine these two attributes, to allow fast, optically sectioned, superresolution imaging. We present a linear reconstruction method that maximizes the axial frequency extent of the combined 2D structured illumination passband along with an empirically optimized approximation to this scheme. These reconstruction methods are compared to other schemes using structured illumination images of fluorescent samples. For sinusoidal excitation at half the incoherent cutoff frequency we find that removing information in the zero order passband except for a small region close to the excitation frequency, where it replaces the complementary information from the displaced first order passband, enables optimal reconstruction of optically sectioned images with enhanced spatial resolution.

Rapid quantitative phase imaging for partially coherent light microscopy

Abstract: Partially coherent light provides promising advantages for imaging applications. In contrast to its completely coherent counterpart, it prevents image degradation due to speckle noise and decreases cross-talk among the imaged objects. These facts make attractive the partially coherent illumination for accurate quantitative imaging in microscopy. In this work, we present a non-interferometric technique and system for quantitative phase imaging with simultaneous determination of the spatial coherence properties of the sample illumination. Its performance is experimentally demonstrated in several examples underlining the benefits of partial coherence for practical imagining applications. The programmable optical setup comprises an electrically tunable lens and sCMOS camera that allows for high-speed measurement in the millisecond range.

Image reconstruction for structured-illumination microscopy with low signal level

Abstract: We report a new image processing technique for the structured illumination microscopy designed to work with low signals, with the goal of reducing photobleaching and phototoxicity of the sample. Using a pre-filtering process to estimate experimental parameters and total variation as a constraint to reconstruct, we obtain two orders of magnitude of exposure reduction while maintaining the resolution improvement and image quality compared to a standard structured illumination microscopy. The algorithm is validated on both fixed and live cell data with results confirming that we can image more than 15x more time points compared to the standard technique.