Full-field Microscopy Research Articles

Bioimaging Using Full Field and Contact EUV and SXR Microscopes with Nanometer Spatial Resolution

We present our recent results, related to nanoscale imaging in the extreme ultraviolet (EUV) and soft X-ray (SXR) spectral ranges and demonstrate three novel imaging systems recently developed for the purpose of obtaining high spatial resolution images of nanoscale objects with the EUV and SXR radiations. All the systems are based on laser-plasma EUV and SXR sources, employing a double stream gas puff target. The EUV and SXR full field microscopes—operating at 13.8 nm and 2.88 nm wavelengths, respectively—are currently capable of imaging nanostructures with a sub-50 nm spatial resolution with relatively short (seconds) exposure times. The third system is a SXR contact microscope, operating in the “water-window” spectral range (2.3–4.4 nm wavelength), to produce an imprint of the internal structure of the investigated object in a thin surface layer of SXR light sensitive poly(methyl methacrylate) photoresist. The development of such compact imaging systems is essential to the new research related to biological science, material science, and nanotechnology applications in the near future. Applications of all the microscopes for studies of biological samples including carcinoma cells, diatoms, and neurons are presented. Details about the sources, the microscopes, as well as the imaging results for various objects will be shown and discussed.

Compact interferometric module for full-field interferometric phase microscopy with low spatial coherence illumination.

We propose a compact and external off-axis interferometric module that can achieve interference with low spatial coherence illumination over the entire field of view. The interferometer is easy to align and stable and can be connected to the output of an existing microscope illuminated with a low spatial coherence light source, thus allowing quantitative phase imaging with a low degree of spatial noise. We demonstrate the imaging and the interference properties of the proposed interferometric module and use it for quantitative phase imaging of reflective samples.

Simulating and optimizing compound refractive lens-based X-ray microscopes.

A comprehensive optical description of compound refractive lenses (CRLs) in condensing and full-field X-ray microscopy applications is presented. The formalism extends ray-transfer matrix analysis by accounting for X-ray attenuation by the lens material. Closed analytical expressions for critical imaging parameters such as numerical aperture, spatial acceptance (vignetting), chromatic aberration and focal length are provided for both thin- and thick-lens imaging geometries. These expressions show that the numerical aperture will be maximized and chromatic aberration will be minimized at the thick-lens limit. This limit may be satisfied by a range of CRL geometries, suggesting alternative approaches to improving the resolution and efficiency of CRLs and X-ray microscopes.

Towards on-the-fly data post-processing for real-time tomographic imaging at TOMCAT

Sub-second full-field tomographic microscopy at third-generation synchrotron sources is a reality, opening up new possibilities for the study of dynamic systems in different fields. Sustained elevated data rates of multiple GB/s in tomographic experiments will become even more common at diffraction-limited storage rings, coming in operation soon. The computational tools necessary for the post-processing of raw tomographic projections have generally not experienced the same efficiency increase as the experimental facilities, hindering optimal exploitation of this new potential. We present here a fast, flexible, and user-friendly post-processing pipeline overcoming this efficiency mismatch and delivering reconstructed tomographic datasets just few seconds after the data have been acquired, enabling fast parameter and image quality evaluation as well as efficient post-processing of TBs of tomographic data. With this new tool, also able to accept a stream of data directly from a detector, few selected tomographic slices are available in less than half a second, providing advanced previewing capabilities paving the way to new concepts for on-the-fly control of dynamic experiments.

Ultrahigh-speed, phase-sensitive full-field interferometric confocal microscopy for quantitative microscale physiology.

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of in vivo microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 μm lateral resolution, ~8 μm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model (Xenopus embryos) with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.

Scanning coherent diffractive imaging methods for actinic extreme ultraviolet mask metrology

For the successful implementation of extreme ultraviolet (EUV) lithography in the upcoming technology nodes, a major challenge to overcome is the stable and reliable detection and characterization of mask defects. We have recently presented a reflective mode EUV mask scanning lensless imaging tool (RESCAN) which was installed at the XIL-II beamline of the swiss light source and showed reconstructed aerial images of test patterns on EUV masks. RESCAN uses scanning coherent diffractive imaging (SCDI) methods to obtain actinic aerial images of EUV photomasks and was designed for 80 nm onmask resolution. Our SCDI algorithm reconstructs the measured sample by iteratively solving the phase problem using overdetermined diffraction data gathered by scanning across the specimen with a finite illumination. It provides the phase and amplitude aerial images of EUV photomasks with high resolution without the need to use high numerical aperture (NA) lenses. Contrary to scanning microscopy and full-field microscopy, where the resolution is limited by the spot size or NA of the lens, the achievable resolution with our method depends on the detector noise and NA of the detector. To increase the resolution of our tool, we upgraded RESCAN with a detector and algorithms. Here, we present the results obtained with the tool that is capable of up to 40-nm onmask resolution. We believe that the realization of our prototype marks a significant step toward overcoming the limitations imposed by methods relying on imaging optics and shows a viable solution for actinic mask metrology.

Elastic instabilities in planar elongational flow of monodisperse polymer solutions

We investigate purely elastic flow instabilities in the almost ideal planar stagnation point elongational flow field generated by a microfluidic optimized-shape cross-slot extensional rheometer (OSCER). We use time-resolved flow velocimetry and full-field birefringence microscopy to study the behavior of a series of well-characterized viscoelastic polymer solutions under conditions of low fluid inertia and over a wide range of imposed deformation rates. At low deformation rates the flow is steady and symmetric and appears Newtonian-like, while at high deformation rates we observe the onset of a flow asymmetry resembling the purely elastic instabilities reported in standard-shaped cross-slot devices. However, for intermediate rates, we observe a new type of elastic instability characterized by a lateral displacement and time-dependent motion of the stagnation point. At the onset of this new instability, we evaluate a well-known dimensionless criterion M that predicts the onset of elastic instabilities based on geometric and rheological scaling parameters. The criterion yields maximum values of M which compare well with critical values of M for the onset of elastic instabilities in viscometric torsional flows. We conclude that the same mechanism of tension acting along curved streamlines governs the onset of elastic instabilities in both extensional (irrotational) and torsional (rotational) viscoelastic flows.

Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

We present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional data cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. We demonstrate this ‘big data’ approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.

Bimodal endoscopic probe combining white-light microscopy and optical coherence tomography.

We present a novel bimodal endoscopic imaging probe that can simultaneously provide full-field white-light video microscopy and confocal optical coherence tomography (OCT) depth scans. The two modalities rely on spectrally separated optical paths that run partially in parallel through a micro-optical bench system, which has a cross-section of only 2 mm×2.76 mm and is realized via standard silicon micromachining techniques. With a numerical aperture of 0.061, the video modality has a resolution and field of view of 9.3 and 1240 μm×1080 μm, respectively. The resolution is limited by the pixel spacing of the coherent fiber bundle, which relays the acquired image from the distal to the proximal end. A custom-designed diffractive optical element placed within the video imaging path significantly improves the image contrast by up to 45% in the medium frequency range. The OCT modality is optimized for 830nm center wavelength, and works in a confocal arrangement with an NA of 0.018. It provides single-point depth probing at the center of the video image with a lateral resolution of 20μm. Through its compact footprint and enhanced functionality, the probe can provide depth-resolved guiding capability for existing laparoscopes and represents a major step toward a new class of multimodal endoscopic imaging probes.

Simulation of aperture-optimised refractive lenses for hard X-ray full field microscopy.

The aperture of refractive X-ray lenses is limited by absorption and geometry. We introduce a specific simulation method to develop an aperture-optimized lens design for hard X-ray full field microscopy. The aperture-optimized lens, referred to as Taille-lens, allows for high spatial resolution as well as homogeneous image quality. This is achieved by the individual adaptation of the apertures of hundreds of lens elements of an X-ray imaging lens to the respective microscopy setup. For full field microscopy, the simulations result in lenses with both a large entrance and exit aperture and lens elements with smaller apertures in the middle of the lens.

Contrast transfer functions for Zernike phase contrast in full-field transmission hard X-ray microscopy

Full-field transmission hard X-ray microscopy (TXM) has been widely applied to study morphology and structures with high spatial precision and to dynamic processes. Zernike phase contrast (ZPC) in hard X-ray TXM is often utilized to get an in-line phase contrast enhancement for weak-absorbing materials with little contrast differences. Here, following forward image formation, we derive and simplify the contrast transfer functions (CTFs) of the Zernike phase imaging system in TXM based on a linear space-shift-invariant imaging mode under certain approximations. The CTFs in ZPC in their simplified forms show a high similarity to the one in free-space propagation X-ray imaging systems.

A feasibility study of full-field X-ray orientation microscopy at the onset of deformation twinning

Three-dimensional X-ray orientation microscopy based on X-ray full-field imaging techniques such as diffraction contrast tomography is a challenging task when it comes to materials displaying non-negligible intragranular orientation spread and/or intricate grain microstructures as a result of plastic deformation and deformation twinning. As shown in this article, the optimization of the experimental conditions and a number of modifications of the data analysis routines enable detection and three-dimensional reconstruction of twin lamellae down to micrometre thickness, as well as more accurate three-dimensional reconstruction of grains displaying intragranular orientation spreads of up to a few degrees. The reconstruction of spatially resolved orientation maps becomes possible through the use of a recently introduced six-dimensional reconstruction framework, which has been further extended in order to enable simultaneous reconstruction of parent and twin orientations and to account for the finite impulse response of the X-ray imaging detector. The simultaneous reconstruction of disjoint orientation domains requires appropriate scaling of the scattering intensities based on structure and Lorentz factors and yields three-dimensional reconstructions with comparable density values for all the grains. This in turn enables the use of a global intensity-guided assembly procedure and avoids problems related to the single-grain thresholding procedure used previously. Last but not least, carrying out a systematic search over the list of known twin variants (forward modelling) for each of the indexed parent grains, it is possible to identify additional twins which have been left undetected at the previous stage of grain indexing based on diffraction spot peak positions. The enhanced procedure has been tested on a 1% deformed specimen made from a Ti–4% Al alloy and the result has been cross-validated against a two-dimensional electron backscatter diffraction orientation map acquired on one of the lateral sample surfaces.

Rapid Mapping of Lithiation Dynamics in Transition Metal Oxide Particles with Operando X-ray Absorption Spectroscopy

Since the commercialization of lithium ion batteries (LIBs), layered transition metal oxides (LiMO2, where M = Co, Mn, Ni, or mixtures thereof) have been materials of choice for LIB cathodes. During cycling, the transition metals change their oxidation states, an effect that can be tracked by detecting energy shifts in the X-ray absorption near edge structure (XANES) spectrum. X-ray absorption spectroscopy (XAS) can therefore be used to visualize and quantify lithiation kinetics in transition metal oxide cathodes; however, in-situ measurements are often constrained by temporal resolution and X-ray dose, necessitating compromises in the electrochemistry cycling conditions used or the materials examined. We report a combined approach to reduce measurement time and X-ray exposure for operando XAS studies of lithium ion batteries. A highly discretized energy resolution coupled with advanced post-processing enables rapid yet reliable identification of the oxidation state. A full-field microscopy setup provides sub-particle resolution over a large area of battery electrode, enabling the oxidation state within many transition metal oxide particles to be tracked simultaneously. Here, we apply this approach to gain insights into the lithiation kinetics of a commercial, mixed-metal oxide cathode material, nickel cobalt aluminium oxide (NCA), during (dis)charge and its degradation during overcharge.

Three-dimensional full-field X-ray orientation microscopy.

A previously introduced mathematical framework for full-field X-ray orientation microscopy is for the first time applied to experimental near-field diffraction data acquired from a polycrystalline sample. Grain by grain tomographic reconstructions using convex optimization and prior knowledge are carried out in a six-dimensional representation of position-orientation space, used for modelling the inverse problem of X-ray orientation imaging. From the 6D reconstruction output we derive 3D orientation maps, which are then assembled into a common sample volume. The obtained 3D orientation map is compared to an EBSD surface map and local misorientations, as well as remaining discrepancies in grain boundary positions are quantified. The new approach replaces the single orientation reconstruction scheme behind X-ray diffraction contrast tomography and extends the applicability of this diffraction imaging technique to material micro-structures exhibiting sub-grains and/or intra-granular orientation spreads of up to a few degrees. As demonstrated on textured sub-regions of the sample, the new framework can be extended to operate on experimental raw data, thereby bypassing the concept of orientation indexation based on diffraction spot peak positions. This new method enables fast, three-dimensional characterization with isotropic spatial resolution, suitable for time-lapse observations of grain microstructures evolving as a function of applied strain or temperature.

Full-field hard x-ray microscopy with interdigitated silicon lenses

Full-field x-ray microscopy using x-ray objectives has become a mainstay of the biological and materials sciences. However, the inefficiency of existing objectives at x-ray energies above 15keV has limited the technique to weakly absorbing or two-dimensional (2D) samples. Here, we show that significant gains in numerical aperture and spatial resolution may be possible at hard x-ray energies by using silicon-based optics comprising ‘interdigitated’ refractive silicon lenslets that alternate their focus between the horizontal and vertical directions. By capitalizing on the nano-manufacturing processes available to silicon, we show that it is possible to overcome the inherent inefficiencies of silicon-based optics and interdigitated geometries. As a proof-of-concept of Si-based interdigitated objectives, we demonstrate a prototype interdigitated lens with a resolution of ≈255nm at 17keV.

Magnetic coupling of vortices in a two-dimensional lattice

We investigated the magnetization reversal of magnetic vortex structures in a two-dimensional lattice. The structures were formed by permalloy (Py) film deposition onto large arrays of self-assembled spherical SiO2-particles with a diameter of 330 nm. We present the dependence of the nucleation and annihilation field of the vortex structures as a function of the Py layer thickness (aspect ratio) and temperature. By increasing the Py thickness up to 90 nm or alternatively by lowering the temperature the vortex structure becomes more stable as expected. However, the increase of the Py thickness results in the onset of strong exchange coupling between neighboring Py caps due to the emergence of Py bridges connecting them. In particular, we studied the influence of magnetic coupling locally by in-field scanning magneto-resistive microscopy and full-field magnetic soft x-ray microscopy, revealing a domain-like nucleation process of vortex states, which arises via domain wall propagation due to exchange coupling of the closely packed structures. By analyzing the rotation sense of the reversed areas, large connected domains are present with the same circulation sense. Furthermore, the lateral core displacements when an in-plane field is applied were investigated, revealing spatially enlarged vortex cores and a broader distribution with increasing Py layer thickness. In addition, the presence of some mixed states, vortices and c-states, is indicated for the array with the thickest Py layer.

Localization-based full-field microscopy: how to attain super-resolved images.

In this study, we have investigated localization-based microscopy to achieve full-field super-resolution. For localized sampling, we have considered combs consisting of unit pulses and near-fields localized by surface nanoapertures. Achievable images after reconstruction were assessed in terms of peak signal-to-noise ratio (PSNR). It was found that spatial switching of individual pulses may be needed to break the diffraction limit. Among the parameters, the resolution was largely determined by sampling period while the effect of width of a sampling pulse on PSNR was relatively limited. For the range of sampling parameters that we considered, the highest resolution achievable is estimated to be 70 nm, which can further be enhanced by optimizing the localization parameters.

Artifact characterization and reduction in scanning X-ray Zernike phase contrast microscopy.

Zernike phase contrast microscopy is a well-established method for imaging specimens with low absorption contrast. It has been successfully implemented in full-field microscopy using visible light and X-rays. In microscopy Cowley's reciprocity principle connects scanning and full-field imaging. Even though the reciprocity in Zernike phase contrast has been discussed by several authors over the past thirty years, only recently it was experimentally verified using scanning X-ray microscopy. In this paper, we investigate the image and contrast formation in scanning Zernike phase contrast microscopy with a particular and detailed focus on the origin of imaging artifacts that are typically associated with Zernike phase contrast. We demonstrate experimentally with X-rays the effect of the phase mask design on the contrast and halo artifacts and present an optimized design of the phase mask with respect to photon efficiency and artifact reduction. Similarly, due to the principle of reciprocity the observations and conclusions of this work have direct applicability to Zernike phase contrast in full-field microscopy as well.

High-axial-resolution, full-field optical coherence microscopy using tungsten halogen lamp and liquid-crystal-based achromatic phase shifter.

A high-axial-resolution, full-field optical coherence microscope (FFOCM) for topography and tomography applications is presented. The FFOCM is based on a polarization Linnik interference microscope equipped with a tungsten halogen lamp. The phase difference between the reference and test beams in the microscope is precisely and quickly shifted by using an achromatic liquid-crystal phase shifter (LCPS). The cross-sectional amplitude and phase maps of an interferogram are retrieved by using a three-step phase-shifting technique. The LCPS consists of three identical nematic liquid-crystal (NLC) cells sandwiched between two quarter-wave plates so that it functions as a typical quarter-half-quarter phase shifter. Instead of using high-cost NLC cells with precise thickness of half-wave retardation, a method is proposed to operate thicker NLC cells without scarifying the axial resolution. Experimental results reveal that the FFOCM is able to perform three-dimensional micrometer-resolution imaging.

Quantitative x-ray magnetic circular dichroism mapping with high spatial resolution full-field magnetic transmission soft x-ray spectro-microscopy

The spectroscopic analysis of X-ray magnetic circular dichroism (XMCD), which serves as strong and element-specific magnetic contrast in full-field magnetic transmission soft x-ray microscopy, is shown to provide information on the local distribution of spin (S) and orbital (L) magnetic moments down to a spatial resolution of 25 nm limited by the x-ray optics used in the x-ray microscope. The spatially resolved L/S ratio observed in a multilayered (Co 0.3 nm/Pt 0.5 nm) × 30 thin film exhibiting a strong perpendicular magnetic anisotropy decreases significantly in the vicinity of domain walls, indicating a non-uniform spin configuration in the vertical profile of a domain wall across the thin film. Quantitative XMCD mapping with x-ray spectro-microscopy will become an important characterization tool for systems with topological or engineered magnetization inhomogeneities.