Selective activation of photoactivatable fluorescent protein based on binary holography.

The ability to deliver laser doses to different target locations with high spatial and temporal resolution has been a long-sought goal in photo-stimulation and optogenetics research via, for example, photoactivatable proteins. These light-sensitive proteins undergo conformational changes upon photoactivation, serving functions such as triggering fluorescence, modulating ion channel activities, or initiating biochemical reactions within cells. Conventionally, photo-stimulation on light-sensitive proteins is performed by serially scanning a laser focus or via 2D projection, which is limited by relatively low spatiotemporal resolution. In this work, we present a programmable two-photon stimulation method based on a digital micromirror device (DMD) and binary holography to perform the activation of photoactivatable green fluorescent protein (PAGFP) in live cells. This method achieved grayscale and 3D selective PAGFP activation with subcellular resolution. In the experiments, we demonstrated the 3D activation capability and investigated the diffusion dynamics of activated PAGFP on the cell membrane. A regional difference in cell membrane diffusivity was observed, indicating the great potential of our approach in interrogating the spatiotemporal dynamics of cellular processes inside living cells.

Compressive sensing-based multi-focus line-scanning two-photon microscopy for fast 3D imaging.

Fast 3D volume imaging methods have been playing increasingly important roles in biological studies. In this article, we present the design and characterization of a multi-focus line-scanning two-photon microscope. Specifically, a digital micromirror device (DMD) is employed to generate a randomly distributed focus array on a plane (i.e., x-z) via binary holography. Next, a galvanometric mirror scans the focus array in a direction normal to the plane (i.e., y-axis) over the imaging volume. For sparse samples, e.g., neural networks in a brain, 1-3 foci are used together with compressive sensing algorithm to achieve a volume imaging rate of 15.5 volumes/sec over 77 × 120 × 40 µm3. High-resolution optical cross-sectional images on selected planes and regions can be generated by sequentially scanning the laser focus generated on the x-z plane with good imaging speeds (e.g., 107 frames/sec over 80 × 120 × 40 µm3). In the experiments, microbeads, pollens, and mouse brain slices have been imaged to characterize the point spread function and volume image rate and quality at different sampling ratios. The results show that the multi-focus line-scanning microscope presents a fast and versatile 3D imaging platform for deep tissue imaging and dynamic live animal studies.

Serotonin Syndrome-Mimicking Manifestations in a Patient with Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a complication of systemic lupus erythematosus with diverse clinical presentations sharing common features with variable neurologic disorders. Magnetic resonance imaging (MRI) may provide imaging evidence of structural brain abnormalities associated with symptoms of NPSLE. Serotonin syndrome is a toxidrome characterized by altered mental status, autonomic hyperactivity, and neuromuscular abnormalities. It is mostly caused by medications that increase serotonin and is rarely reported as a manifestation of neuropsychiatric lupus. We presented the case of a 24-year-old Taiwanese woman with a history of systemic lupus erythematosus diagnosed at 21 years of age. The initial clinical and laboratory presentations upon diagnosis included fever, arthritis, hypocomplementemia, positive antinuclear antibody, anti-double-stranded DNA antibody, and anti-ribosomal P antibody. Her condition once remained stable under oral glucocorticoids and immunosuppressants, but she developed sudden-onset consciousness disturbance, incoherent speech, and unsteady gait ten days before our assessment. A high fever of up to 39 °C with tremor and clonus occurred at the intensive care unit. Brain MRI revealed symmetric T2 hyperintensity without diffusion restriction over the bilateral globus pallidus. High-dose pulse glucocorticoid and rituximab were prescribed during her admission and the neuropsychiatric symptoms diminished upon treatment. No alternation in mental status or involuntary movements were noted at follow-up. Our patient was diagnosed with neuropsychiatric lupus, with clinical symptoms and image findings mimicking those of serotonin syndrome. Neuroimaging, such as MRI, detects various structural brain abnormalities and may provide pathophysiological evidence of clinical manifestations.

Scan-less 3D microscopy based on spatiotemporal encoding on a single-cavity dual-comb laser.

Dual-comb microscopy enables high-speed and high-precision optical sampling by simultaneously extracting both amplitude and phase information from the interference signals with frequency division multiplexing. In this Letter, we introduce a spatiotemporal encoding approach for dual-comb microscopy that overcomes previous limitations such as mechanical scanning, low sampling efficiency, and system complexity. By employing free-space angular-chirp-enhanced delay (FACED) and a low-noise single-cavity dual-comb laser, we achieve scan-less 3D imaging with nanometer precision and a 3D distance-imaging rate of 330 Hz, restricted only by the repetition rate difference of the dual-comb laser. Specifically, the FACED unit linearly arranges the laser beam into an array. A grating subsequently disperses this array transversely into lines, facilitating ultrafast spectroscopic applications that are 1-2 orders of magnitude quicker than traditional dual-comb methods. This spatiotemporal encoding also eases the stringent conditions on various dual-comb laser parameters, such as repetition rates, coherence, and stability. Through carefully designed experiments, we demonstrate that our scan-less system can measure 3D profiles of microfabricated structures at a rate of 7 million pixels per second. Our method significantly enhances measurement speed while maintaining high precision, using a compact light source. This advancement has the potential for broad applications, including phase imaging, surface topography, distance ranging, and spectroscopy.

Pixel-reassigned line-scanning microscopy for fast volumetric super-resolution imaging.

Super-resolution microscopy has revolutionized the field of biophotonics by revealing detailed 3D biological structures. Nonetheless, the technique is still largely limited by the low throughput and hampered by increased background signals for dense or thick biological specimens. In this paper, we present a pixel-reassigned continuous line-scanning microscope for large-scale high-speed 3D super-resolution imaging, which achieves an imaging resolution of 0.41 µm in the lateral direction, i.e., a 2× resolution enhancement from the raw images. Specifically, the recorded line images are first reassigned to the line-excitation center at each scanning position to enhance the resolution. Next, a modified HiLo algorithm is applied to reduce the background signals. Parametric models have been developed to simulate the imaging results of randomly distributed fluorescent beads. Imaging experiments were designed and performed to verify the predicted performance on various biological samples, which demonstrated an imaging speed of 3400 pixels/ms on millimeter-scale specimens. These results confirm the pixel-reassigned line-scanning microscopy is a facile and powerful method to realize large-area super-resolution imaging on thick or dense biological samples.

A cellular resolution atlas of Broca's area.

Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)-referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca's area in a human postmortem specimen, within a whole-brain reference space atlas.

Quasi-2D Dion-Jacobson phase perovskites as a promising material platform for stable and high-performance lasers.

Metal halide perovskites have shown outstanding optoelectronic and nonlinear optical properties; yet, to realize wafer-scale high-performance perovskite-integrated photonics, the materials also need to have excellent ambient stability and compatibility with nanofabrication processes. In this work, we introduce Dion-Jacobson (D-J) phase perovskites for photonic device applications. By combining self-assembled monolayer-assisted film growth with thermal pressing, we obtain a series of compact and extremely smooth D-J phase perovskite thin films that exhibit excellent stability during electron-beam lithography, solvent development, and rinse. Combining spectroscopic and morphological characterizations, we further demonstrate how organic spacers can be used to fine-tune the photophysical properties and processability of the perovskite films. The distributed-feedback lasers based on the D-J phase perovskites exhibit a low lasing threshold (5.5 µJ cm-2 pumped with nanosecond laser), record high Q factor (up to 30,000), and excellent stability, with an unencapsulated device demonstrating a T90 beyond 60 hours in ambient conditions (50% relative humidity).

A survey on the mechanical design for piezo-actuated compliant micro-positioning stages.

This paper presents a comprehensive review of mechanical design and synthesis methods for piezo-actuated compliant micro-positioning stages, which play an important role in areas where high precision motion is required, including bio-robotics, precision manufacturing, automation, and aerospace. Unlike conventional rigid-link mechanisms, the motion of compliant mechanisms is realized by using flexible elements, whereby deformation requires no lubrication while achieving high movement accuracy without friction. As compliant mechanisms differ significantly from traditional rigid mechanisms, recent research has focused on investigating various technologies and approaches to address challenges in the flexure-based micro-positioning stage in the aspects of synthesis, analysis, material, fabrication, and actuation. In this paper, we reviewed the main concepts and key advances in the mechanical design of compliant piezo-actuated micro-positioning stages, with a particular focus on flexure design, kineto-static modeling, actuators, material selection, and functional mechanisms including amplification and self-guiding ones. We also identified the key issues and directions for the development trends of compliant micro-positioning stages.

Multi-Scale Label-Free Human Brain Imaging with Integrated Serial Sectioning Polarization Sensitive Optical Coherence Tomography and Two-Photon Microscopy.

The study of aging and neurodegenerative processes in the human brain requires a comprehensive understanding of cytoarchitectonic, myeloarchitectonic, and vascular structures. Recent computational advances have enabled volumetric reconstruction of the human brain using thousands of stained slices, however, tissue distortions and loss resulting from standard histological processing have hindered deformation-free reconstruction. Here, the authors describe an integrated serial sectioning polarization-sensitive optical coherence tomography (PSOCT) and two photon microscopy (2PM) system to provide label-free multi-contrast imaging of intact brain structures, including scattering, birefringence, and autofluorescence of human brain tissue. The authors demonstrate high-throughput reconstruction of 4 × 4 × 2cm3 sample blocks and simple registration between PSOCT and 2PM images that enable comprehensive analysis of myelin content, vascular structure, and cellular information. The high-resolution 2PM images provide microscopic validation and enrichment of the cellular information provided by the PSOCT optical properties on the same sample, revealing the densely packed fibers, capillaries, and lipofuscin-filled cell bodies in the cortex and white matter. It is  shown that the imaging system enables quantitative characterization of various pathological features in aging process, including myelin degradation, lipofuscin accumulation, and microvascular changes, which opens up numerous opportunities in the study of neurodegenerative diseases in the future.

Multi-Scale Label-free Human Brain Imaging with Integrated Serial Sectioning Polarization Sensitive Optical Coherence Tomography and Two-Photon Microscopy.

The study of neurodegenerative processes in the human brain requires a comprehensive understanding of cytoarchitectonic, myeloarchitectonic, and vascular structures. Recent computational advances have enabled volumetric reconstruction of the human brain using thousands of stained slices, however, tissue distortions and loss resulting from standard histological processing have hindered deformation-free reconstruction of the human brain. The development of a multi-scale and volumetric human brain imaging technique that can measure intact brain structure would be a major technical advance. Here, we describe the development of integrated serial sectioning Polarization Sensitive Optical Coherence Tomography (PSOCT) and Two Photon Microscopy (2PM) to provide label-free multi-contrast imaging, including scattering, birefringence and autofluorescence of human brain tissue. We demonstrate that high-throughput reconstruction of 4×4×2cm3 sample blocks and simple registration of PSOCT and 2PM images enable comprehensive analysis of myelin content, vascular structure, and cellular information. We show that 2µm in-plane resolution 2PM images provide microscopic validation and enrichment of the cellular information provided by the PSOCT optical property maps on the same sample, revealing the sophisticated capillary networks and lipofuscin filled cell bodies across the cortical layers. Our method is applicable to the study of a variety of pathological processes, including demyelination, cell loss, and microvascular changes in neurodegenerative diseases such as Alzheimer's disease (AD) and Chronic Traumatic Encephalopathy (CTE).

Parallel two-photon lithography achieving uniform sub-200†nm features with thousands of individually controlled foci.

The limited throughput of nano-scale laser lithography has been the bottleneck for its industrial applications. Although using multiple laser foci to parallelize the lithography process is an effective and straightforward strategy to improve rate, most conventional multi-focus methods are plagued by non-uniform laser intensity distribution due to the lack of individual control for each focus, which greatly hinders the nano-scale precision. In this paper, we present a highly uniform parallel two-photon lithography method based on a digital mirror device (DMD) and microlens array (MLA), which allows the generation of thousands of femtosecond (fs) laser foci with individual on-off switching and intensity-tuning capability. In the experiments, we generated a 1,600-laser focus array for parallel fabrication. Notably, the intensity uniformity of the focus array reached 97.7%, where the intensity-tuning precision for each focus reached 0.83%. A uniform dot array structure was fabricated to demonstrate parallel fabrication of sub-diffraction limit features, i.e., below 1/4 λ or 200 nm. The multi-focus lithography method has the potential of realizing rapid fabrication of sub-diffraction, arbitrarily complex, and large-scale 3D structures with three orders of magnitude higher fabrication rate.

Ultrafast 3D nanofabrication via digital holography.

There has been a compelling demand of fabricating high-resolution complex three-dimensional (3D) structures in nanotechnology. While two-photon lithography (TPL) largely satisfies the need since its introduction, its low writing speed and high cost make it impractical for many large-scale applications. We report a digital holography-based TPL platform that realizes parallel printing with up to 2000 individually programmable laser foci to fabricate complex 3D structures with 90 nm resolution. This effectively improves the fabrication rate to 2,000,000 voxels/sec. The promising result is enabled by the polymerization kinetics under a low-repetition-rate regenerative laser amplifier, where the smallest features are defined via a single laser pulse at 1 kHz. We have fabricated large-scale metastructures and optical devices of up to centimeter-scale to validate the predicted writing speed, resolution, and cost. The results confirm our method provides an effective solution for scaling up TPL for applications beyond laboratory prototyping.

Three-dimensional nanofabrication via ultrafast laser patterning and kinetically regulated material assembly.

A major challenge in nanotechnology is the fabrication of complex three-dimensional (3D) structures with desired materials. We present a strategy for fabricating arbitrary 3D nanostructures with a library of materials including metals, metal alloys, 2D materials, oxides, diamond, upconversion materials, semiconductors, polymers, biomaterials, molecular crystals, and inks. Specifically, hydrogels patterned by femtosecond light sheets are used as templates that allow for direct assembly of materials to form designed nanostructures. By fine-tuning the exposure strategy and features of the patterned gel, 2D and 3D structures of 20- to 200-nm resolution are realized. We fabricated nanodevices, including encrypted optical storage and microelectrodes, to demonstrate their designed functionality and precision. These results show that our method provides a systematic solution for nanofabrication across different classes of materials and opens up further possibilities for the design of sophisticated nanodevices.

Diverse and Composite Roles of miRNA in Non-Neuronal Cells and Neuronal Synapses in Alzheimer's Disease.

Neurons interact with astrocytes, microglia, and vascular cells. These interactions become unbalanced in disease states, resulting in damage to neurons and synapses, and contributing to cognitive impairment. Importantly, synaptic loss and synaptic dysfunction have been considered for years as a main pathological factor of cognitive impairment in Alzheimer's disease (AD). Recently, miRNAs have emerged as essential regulators of physiological and pathological processes in the brain. Focusing on the role of miRNAs in regulating synaptic functions, as well as different cell types in the brain, offers opportunities for the early prevention, diagnosis, and potential treatment of AD-related cognitive impairment. Here, we review the recent research conducted on miRNAs regulating astrocytes, microglia, cerebrovasculature, and synaptic functions in the context of AD-related cognitive impairment. We also review potential miRNA-related biomarkers and therapeutics, as well as emerging imaging technologies relevant for AD research.

Main Bioactive Components and Their Biological Activities from Natural and Processed Rhizomes of Polygonum sibiricum.

Polygonatum sibiricum (Asparagaceae) is often used as an herbal drug in the traditional medicine of Southeast Asia. Its rhizome, called "Huang Jing", is used in traditional Chinese medicine as an immune system stimulant, hypolipidemic agent, anti-aging agent, anti-fatigue agent, and cardiovascular protectant. We investigated the antioxidant, anti-acetylcholinesterase (AChE), anti-inflammatory, and anti-α-glucosidase effects of various solvent extracts and major bioactive components of Polygonatum sibiricum (PS) and processed Polygonatum sibiricum (PPS). Dichloromethane extract of PS showed stronger antioxidant effects by DPPH, ABTS, and FRAP assays, and EtOAc extract displayed relatively high antioxidant activity by a superoxide radical scavenging test. Moreover, acetone, EtOAc, and dichloromethane extracts displayed a significant anti-α-glucosidase effect. EtOH and CH2Cl2 extracts showed effective AChE inhibitory activity. In addition, dichloromethane extract showed the best inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) accumulation in RAW264.7 macrophages. HPLC analysis was used to investigate and compare the content of major active components of various solvent extracts of PS and PPS. Rutin showed the most effective scavenging of DPPH and ABTS free radicals, while scopoletin and isoquercetin displayed the strongest anti-α-glucosidase and anti-AChE effect, respectively. Rutin showed the best inhibition against LPS-induced NO production and also inhibited inducible nitric oxide synthase (iNOS) expression in Western blot. The molecular docking of AChE and iNOS revealed that active components could have a better antagonistic effect than positive controls (common inhibitors). This study shows that the active extracts and components of Polygonatum sibiricum have the potential to be further developed as a natural anti-AChE, anti-α-glucosidase, antioxidant and anti-inflammatory agent.

Optimal nonlinear Stokes-Mueller polarimetry for multi-photon processes.

In this Letter, we present an optimization model for nonlinear Stokes-Mueller polarimetry (SMP) to improve the precision in estimating the nonlinear Mueller matrix (MM) for two- and three-photon processes. Although nonlinear polarimeters can measure the polarization properties of multi-photon processes or materials, existing methods are suboptimal, leading to low measurement precision. Based on the model and its solution, we have designed a new measurement strategy to substantially reduce the estimation variance of nonlinear MM coefficients by approximately 58.2% for second-harmonic generation polarimetry and 78.7% for third-harmonic generation polarimetry. The model and measurement method can be directly applied to multi-photon processes to improve the precision of SMP.

Underwater image restoration via Stokes decomposition.

In this Letter, we present a Stokes imaging-based method to restore objects and enhance image contrast in turbid water. In the system, a light source illuminates the objects with two orthometric polarization states; based on a new Stokes decomposition model, the recorded images are converted to Stokes maps and subsequently restored to a clear image, free of reflections and scattered lights. A mathematical model has been developed to explain the Stokes decomposition and how the undesired reflections and scattered lights are rejected. Imaging experiments have been devised and performed on different objects, e.g., metals and plastics, under different turbidities. The results demonstrate enhanced image quality and capability to distinguish polarization differences. This new, to the best of our knowledge, method can be readily applied to practical underwater object detection and potentially realize clear vision in other scattering media.

Volumetric Characterization of Microvasculature in Ex Vivo Human Brain Samples By Serial Sectioning Optical Coherence Tomography.

OBJECTIVE: Serial sectioning optical coherence tomography (OCT) enables accurate volumetric reconstruction of several cubic centimeters of human brain samples. We aimed to identify anatomical features of the ex vivo human brain, such as intraparenchymal blood vessels and axonal fiber bundles, from the OCT data in 3D, using intrinsic optical contrast. METHODS: We developed an automatic processing pipeline to enable characterization of the intraparenchymal microvascular network in human brain samples. RESULTS: We demonstrated the automatic extraction of the vessels down to a 20 µm in diameter using a filtering strategy followed by a graphing representation and characterization of the geometrical properties of microvascular network in 3D. We also showed the ability to extend this processing strategy to extract axonal fiber bundles from the volumetric OCT image. CONCLUSION: This method provides a viable tool for quantitative characterization of volumetric microvascular network as well as the axonal bundle properties in normal and pathological tissues of the ex vivo human brain.

Self-calibration for Mueller polarimeters based on DoFP polarization imagers.

Mueller polarimeters (MPs) based on division of focal plane (DoFP) polarization imagers can achieve fast measurements and significantly improve the effectiveness of Mueller polarimetry. In this Letter, we demonstrate a unique property of the DoFP sensor-based MPs: they can be calibrated without any extra polarizing reference element. We describe a self-calibration method that only requires six image acquisitions; based on our analysis, the calibration accuracy is only limited by the noise.

Enhanced polarization demosaicking network via a precise angle of polarization loss calculation method.

Many existing polarization networks reconstruct polarization information based on calculating the angle of polarization (AoP) loss. Yet, the conventional loss calculation method, which is based on a linear difference approach, compromises the reconstruction accuracy and causes additional training time when combined with learning-based methods. In this Letter, we present a new, to the best of our knowledge, method to calculate the AoP loss and apply it in an enhanced color polarization demosaicking network with a "multi-branch" structure, i.e., ePDNet. Experiments are performed to demonstrate the efficacy and superiority of the method, which improves the network convergence speed by three times as well as the output image quality. The new method may find important applications in the field of polarimetric imaging.