Aberration Correction for Large-Angle Illumination Scanning Transmission Electron Microscopy by Using Iterative Electron Ptychography Algorithms.

Modern aberration correctors in the scanning transmission electron microscope (STEM) have dramatically improved the attainable spatial resolution and enabled atomical structure and spectroscopic analysis even at low acceleration voltages (≤80 kV). For a large-angle illumination, achieving successful aberration correction to high angles is challenging with an aberration corrector, which limits further improvements in applications such as super-resolution, three-dimensional atomic depth resolution, or atomic surface morphology analyses. Electron ptychography based on four-dimensional STEM can provide a postprocessing strategy to overcome the current technological limitations. In this work, we have demonstrated that aberration correction for large-angle illumination is feasible by pushing the capabilities of regularized ptychographic iterative engine algorithms to reconstruct 4D data sets acquired using a relatively low-efficiency complementary metal oxide semiconductor camera. We report super resolution (0.71 Å) with large-angle illumination (50-60 mrad) and under 60 kV accelerating voltage.

High-resolution open-top axially swept light sheet microscopy.

BACKGROUND: Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for the high-throughput cellular imaging of optically cleared, large-sized specimens, such as the brain. Despite the development of various OT-LSM techniques, achieving submicron resolution in all dimensions remains. RESULTS: We developed a high-resolution open-top axially swept LSM (HR-OTAS-LSM) for high-throughput and high-resolution imaging in all dimensions. High axial and lateral resolutions were achieved by using an aberration-corrected axially swept excitation light sheet in the illumination arm and a high numerical aperture (NA) immersion objective lens in the imaging arm, respectively. The high-resolution, high-throughput visualization of neuronal networks in mouse brain and retina specimens validated the performance of HR-OTAS-LSM. CONCLUSIONS: The proposed HR-OTAS-LSM method represents a significant advancement in the high-resolution mapping of cellular networks in biological systems such as the brain and retina.

Iterative Pulse-Echo Tomography for Ultrasonic Image Correction.

Acoustic aberration, caused by the uneven distribution of tissue speed-of-sound (SoS), significantly reduces the quality of ultrasound imaging. An important approach to mitigate this issue is imaging correction based on local SoS estimation. Computed ultrasound tomography in echo mode (CUTE) is an SoS estimation method that utilizes phase-shift information from ultrasound pulse-echo signals, offering both practical utility and computational efficiency. However, the traditional single-pass CUTE often suffers from poor accuracy and robustness. In this paper, an advanced approach known as iterative CUTE is introduced, which refines SoS estimates through iterative correction of errors and noise, addressing the limitations of traditional single-pass methods. It is argued that traditional precision indicators like root mean square error (RMSE) fall short in adequately reflecting the quality of SoS estimates for imaging correction, and coherence factor (CF) is proposed as a more indicative metric. Performance validation of the iterative CUTE algorithm was conducted using a simulation and agar phantom experiment. The results indicated that the iterative CUTE approach surpasses the single-pass approach, enhancing the average CF for SoS estimates by up to 18.2%. In phantom experiments, imaging corrected with SoS estimates from iterative CUTE reduced the Array Performance Index (API) by up to 40% compared to traditional methods.

Fourier Ptychographic Neural Network Combined with Zernike Aberration Recovery and Wirtinger Flow Optimization.

Fourier ptychographic microscopy, as a computational imaging method, can reconstruct high-resolution images but suffers optical aberration, which affects its imaging quality. For this reason, this paper proposes a network model for simulating the forward imaging process in the Tensorflow framework using samples and coherent transfer functions as the input. The proposed model improves the introduced Wirtinger flow algorithm, retains the central idea, simplifies the calculation process, and optimizes the update through back propagation. In addition, Zernike polynomials are used to accurately estimate aberration. The simulation and experimental results show that this method can effectively improve the accuracy of aberration correction, maintain good correction performance under complex scenes, and reduce the influence of optical aberration on imaging quality.

K-Space Approach in Optical Coherence Tomography: Rigorous Digital Transformation of Arbitrary-Shape Beams, Aberration Elimination and Super-Refocusing beyond Conventional Phase Correction Procedures.

For the most popular method of scan formation in Optical Coherence Tomography (OCT) based on plane-parallel scanning of the illuminating beam, we present a compact but rigorous K-space description in which the spectral representation is used to describe both the axial and lateral structure of the illuminating/received OCT signals. Along with the majority of descriptions of OCT-image formation, the discussed approach relies on the basic principle of OCT operation, in which ballistic backscattering of the illuminating light is assumed. This single-scattering assumption is the main limitation, whereas in other aspects, the presented approach is rather general. In particular, it is applicable to arbitrary beam shapes without the need for paraxial approximation or the assumption of Gaussian beams. The main result of this study is the use of the proposed K-space description to analytically derive a filtering function that allows one to digitally transform the initial 3D set of complex-valued OCT data into a desired (target) dataset of a rather general form. An essential feature of the proposed filtering procedures is the utilization of both phase and amplitude transformations, unlike conventionally discussed phase-only transformations. To illustrate the efficiency and generality of the proposed filtering function, the latter is applied to the mutual transformation of non-Gaussian beams and to the digital elimination of arbitrary aberrations at the illuminating/receiving aperture. As another example, in addition to the conventionally discussed digital refocusing enabling depth-independent lateral resolution the same as in the physical focus, we use the derived filtering function to perform digital "super-refocusing." The latter does not yet overcome the diffraction limit but readily enables lateral resolution several times better than in the initial physical focus.

Mitigating challenges in aberration-corrected electron-beam lithography on electron-opaque substrates.

Aberration-corrected electron-beam lithography (AC-EBL) using ultra-thin electron transparent membranes has achieved single-digit nanometer resolution in two widely used electron-beam resists: poly (methyl methacrylate) (PMMA) and hydrogen silsesquioxane. On the other hand, AC-EBL implementation on thick, electron-opaque substrates is appealing for conventional top-down fabrication of quantum devices with nanometer-scale features. To investigate the performance of AC-EBL on thick substrates, we measured the lithographic point spread function of a 200 keV aberration-corrected scanning transmission electron microscope by defining both positive and negative patterns in PMMA thin films, spin-cast on thick SiO2/Si substrates. We present the problems encountered during pre-exposure beam focusing and discuss methods to overcome them. In addition, applying some of these methods using commercial 50 nm thick SiNXmembranes with thick Si support frames, we printed arrays of holes in PMMA with pitches around 26 nm on SiNX/Si substrates with increasing Si thickness. Our results show that proximity effects from even 50 nm thick SiNXmembranes limit hole arrays to 20 nm pitch; however, down to this limit, the effect of the substrate thickness on the pattern quality is minimal. These results highlight the need for novel resists less susceptible to proximity effects, or resists which can be used directly, after development, as the dielectric material in periodic gates in 2D quantum devices.

Aberration correction in abdominal histotripsy.

In transabdominal histotripsy, ultrasound pulses are focused on the body to noninvasively destroy soft tissues via cavitation. However, the ability to focus is limited by phase aberration, or decorrelation of the ultrasound pulses due to spatial variation in the speed of sound throughout heterogeneous tissue. Phase aberration shifts, broadens, and weakens the focus, thereby reducing the safety and efficacy of histotripsy therapy. This paper reviews and discusses aberration effects in histotripsy and in related therapeutic ultrasound techniques (e.g., high intensity focused ultrasound), with an emphasis on aberration by soft tissues. Methods for aberration correction are reviewed and can be classified into two groups: model-based methods, which use segmented images of the tissue as input to an acoustic propagation model to predict and compensate phase differences, and signal-based methods, which use a receive-capable therapy array to detect phase differences by sensing acoustic signals backpropagating from the focus. The relative advantages and disadvantages of both groups of methods are discussed. Importantly, model-based methods can correct focal shift, while signal-based methods can restore substantial focal pressure, suggesting that both methods should be combined in a 2-step approach. Aberration correction will be critical to improving histotripsy treatments and expanding the histotripsy treatment envelope to enable non-invasive, non-thermal histotripsy therapy for more patients.

Design of Electrostatic Aberration Correctors for Scanning Transmission Electron Microscopy.

In a scanning transmission electron microscope (STEM), producing a high-resolution image generally requires an electron beam focused to the smallest point possible. However, the magnetic lenses used to focus the beam are unavoidably imperfect, introducing aberrations that limit resolution. Modern STEMs overcome this by using hardware aberration correctors comprised of many multipole elements, but these devices are complex, expensive, and can be difficult to tune. We demonstrate a design for an electrostatic phase plate that can act as an aberration corrector. The corrector is comprised of annular segments, each of which is an independent two-terminal device that can apply a constant or ramped phase shift to a portion of the electron beam. We show the improvement in image resolution using an electrostatic corrector. Engineering criteria impose that much of the beam within the probe-forming aperture be blocked by support bars, leading to large probe tails for the corrected probe that sample the specimen beyond the central lobe. We also show how this device can be used to create other STEM beam profiles such as vortex beams and probes with a high degree of phase diversity, which improve information transfer in ptychographic reconstructions.

An Automatic Calibration Method for the Field of View Aberration in a Risley-Prism-Based Image Sensor.

Risley-prism-based image sensors can expand the imaging field of view through beam control. The larger the top angle of the prism, the higher the magnification of the field of view, but at the same time, it aggravates the problem of imaging aberrations, which also puts higher requirements on the aberration correction method for the Risley-prism-based image sensor. To improve the speed, accuracy, and stability of the aberration correction process, an automatic calibration method for the Risley-prism-based image sensor is proposed based on a two-axis turntable. The image datasets of the calibration plate with different prism rotation angles and object distances are acquired using a two-axis turntable. Then, the images of the calibration plate are pre-processed using the bicubic interpolation algorithm. The calibration parameters are finally calculated, and parameter optimization is performed. The experimental results verify the feasibility of this automated calibration method. The reprojection error of the calibration is within 0.26 pixels when the distance of the imaging sensor is 3.6 m from the object, and the fine aberration correction results are observed.

Atmospheric Turbulence Aberration Correction Based on Deep Learning Wavefront Sensing.

In this paper, research was conducted on Deep Learning Wavefront Sensing (DLWS) neural networks using simulated atmospheric turbulence datasets, and a novel DLWS was proposed based on attention mechanisms and Convolutional Neural Networks (CNNs). The study encompassed both indoor experiments and kilometer-range laser transmission experiments employing DLWS. In terms of indoor experiments, data were collected and training was performed on the platform built by us. Subsequent comparative experiments with the Shack-Hartmann Wavefront Sensing (SHWS) method revealed that our DLWS model achieved accuracy on par with SHWS. For the kilometer-scale experiments, we directly applied the DLWS model obtained from the indoor platform, eliminating the need for new data collection or additional training. The DLWS predicts the wavefront from the beacon light PSF in real time and then uses it for aberration correction of the emitted laser. The results demonstrate a substantial improvement in the average peak intensity of the light spot at the target position after closed-loop correction, with a remarkable increase of 5.35 times compared to the open-loop configuration.

Adaptive Ultrasound Focusing Through the Cranial Bone for Non-invasive Treatment of Brain Disorders.

Focused ultrasound holds great promise in therapy for its ability to target non-invasively deep seated tissues with non-ionizing therapeutic beams. Nevertheless, brain applications have been hampered for decades by the presence of the skull. The skull indeed strongly reflects, refracts and absorbs ultrasound, which defocuses the therapeutic ultrasound beams. In this chapter, we will first show how the structure of the skull impacts the ultrasound beams and how it narrows the frequency range that can be envisioned for transcranial therapy. We will then introduce different methods that have been developed and optimized to compensate the defocusing effect of the bone. Finally, we will provide an overview of past, current and future treatments of brain disorders.

X-ray Spectrometry in the Era of Aberration-Corrected Electron Optical Beam Lines.

Aberration correction in the analytical transmission electron microscope is most closely associated with improvements in high-resolution imaging. In this paper, the combination of that technology with new system designs, which optimize both electron optics and x-ray detection, is shown to provide more than a tenfold increase in performance over the last 25 years.

Phase Target-Based Calibration of Projector Radial Chromatic Aberration for Color Fringe 3D Measurement Systems.

The camera and projector are indispensable hardware parts of a color fringe projection 3D measurement system. Chromatic aberration between different color channels of the projector and camera has an impact on the measurement accuracy of the color fringe projection 3D profile measurement. There are many studies on camera calibration, but the chromatic aberration of the projector remains a question deserving of further investigation. In view of the complex system architecture and theoretical derivation of the traditional projector radial chromatic aberration method, a phase target based on projector radial chromatic aberration measurement and the correction method are proposed in this paper. This method uses a liquid crystal display with a holographic projection film as the phase target. The liquid crystal display sequentially displays red, green, and blue horizontal and vertical sinusoidal fringe images. The projector projects red, green, and blue horizontal and vertical sinusoidal fringe images to the phase target in turn, and calculates the absolute phases of the display fringes and reflection fringes, respectively. Taking the green channel as the reference channel, a phase coordinate system is established based on the phases of the vertical and horizontal directions displayed on the display screen, using the phase of the reflection fringes on the display screen as the ideal phase value of the phase point. Then, the phase coordinate system of the red and blue channels is transferred to the green phase coordinate system to calculate the chromatic aberration of the red-green channels and the blue-green channels, and pre-compensation is conducted. Experimental results prove that this method can measure and calibrate the radial chromatic aberration of the projector without being affected by the image quality of the camera. The correction effect of this method is that the maximum chromatic aberration of the red-green channel decreases from 1.9591/pixel to 0.5759/pixel, and the average chromatic aberration decreases from 0.2555/pixel to 0.1865/pixel. In addition, blue-green channel maximum chromatic aberration decreased from 1.8906/pixel to 0.5938/pixel, and the average chromatic aberration decreased from 0.2347/pixel to 0.1907/pixel. This method can improve the projection quality for fringe projection 3D profile measurement technology.

Nanofocusing with aberration-corrected rotationally parabolic refractive X-ray lenses. Corrigendum.

A correction in the paper by Seiboth et al. [(2018). J. Synchrotron Rad. 25, 108-115] is made.

A Do-It-Yourself Hyperspectral Imager Brought to Practice with Open-Source Python.

Commercial hyperspectral imagers (HSIs) are expensive and thus unobtainable for large audiences or research groups with low funding. In this study, we used an existing do-it-yourself push-broom HSI design for which we provide software to correct for spectral smile aberration without using an optical laboratory. The software also corrects an aberration which we call tilt. The tilt is specific for the particular imager design used, but correcting it may be beneficial for other similar devices. The tilt and spectral smile were reduced to zero in terms of used metrics. The software artifact is available as an open-source Github repository. We also present improved casing for the imager design, and, for those readers interested in building their own HSI, we provide print-ready and modifiable versions of the 3D-models required in manufacturing the imager. To our best knowledge, solving the spectral smile correction problem without an optical laboratory has not been previously reported. This study re-solved the problem with simpler and cheaper tools than those commonly utilized. We hope that this study will promote easier access to hyperspectral imaging for all audiences regardless of their financial status and availability of an optical laboratory.

Hard X-ray wavefront correction via refractive phase plates made by additive and subtractive fabrication techniques.

Modern subtractive and additive manufacturing techniques present new avenues for X-ray optics with complex shapes and patterns. Refractive phase plates acting as glasses for X-ray optics have been fabricated, and spherical aberration in refractive X-ray lenses made from beryllium has been successfully corrected. A diamond phase plate made by femtosecond laser ablation was found to improve the Strehl ratio of a lens stack with a numerical aperture (NA) of 0.88 × 10-3 at 8.2 keV from 0.1 to 0.7. A polymer phase plate made by additive printing achieved an increase in the Strehl ratio of a lens stack at 35 keV with NA of 0.18 × 10-3 from 0.15 to 0.89, demonstrating diffraction-limited nanofocusing at high X-ray energies.

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies.

Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Imaging mRNA and protein interactions within neurons.

RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.

Optimal STEM Convergence Angle Selection Using a Convolutional Neural Network and the Strehl Ratio.

The selection of the correct convergence angle is essential for achieving the highest resolution imaging in scanning transmission electron microscopy (STEM). The use of poor heuristics, such as Rayleigh's quarter-phase rule, to assess probe quality and uncertainties in the measurement of the aberration function results in the incorrect selection of convergence angles and lower resolution. Here, we show that the Strehl ratio provides an accurate and efficient way to calculate criteria for evaluating the probe size for STEM. A convolutional neural network trained on the Strehl ratio is shown to outperform experienced microscopists at selecting a convergence angle from a single electron Ronchigram using simulated datasets. Generating tens of thousands of simulated Ronchigram examples, the network is trained to select convergence angles yielding probes on average 85% nearer to optimal size at millisecond speeds (0.02% of human assessment time). Qualitative assessment on experimental Ronchigrams with intentionally introduced aberrations suggests that trends in the optimal convergence angle size are well modeled but high accuracy requires a high number of training datasets. This near-immediate assessment of Ronchigrams using the Strehl ratio and machine learning highlights a viable path toward the rapid, automated alignment of aberration-corrected electron microscopes.

A Single Far-Field Deep Learning Adaptive Optics System Based on Four-Quadrant Discrete Phase Modulation.

In adaptive optics (AO), multiple different incident wavefronts correspond to a same far-field intensity distribution, which leads to a many-to-one mapping. To solve this problem, a single far-field deep learning adaptive optics system based on four-quadrant discrete phase modulation (FQDPM) is proposed. Our method performs FQDPM on an incident wavefront to overcome this many-to-one mapping, then convolutional neural network (CNN) is used to directly predict the wavefront. Numerical simulations indicate that the proposed method can achieve precise high-speed wavefront correction with a single far-field intensity distribution: it takes nearly 0.6ms to complete wavefront correction while the mean root mean square (RMS) of residual wavefronts is 6.3% of that of incident wavefronts, and the Strehl ratio of the far-field intensity distribution increases by 5.7 times after correction. In addition, the experiment results show that mean RMS of residual wavefronts is 6.5% of that of incident wavefronts and it takes nearly 0.5 ms to finish wavefront reconstruction, which verifies the correctness of our proposed method.