The ultra-thin, minimally invasive surface electrode array NeuroWeb for probing neural activity.

Electrophysiological recording technologies can provide valuable insights into the functioning of the central and peripheral nervous systems. Surface electrode arrays made of soft materials or implantable multi-electrode arrays with high electrode density have been widely utilized as neural probes. However, neither of these probe types can simultaneously achieve minimal invasiveness and robust neural signal detection. Here, we present an ultra-thin, minimally invasive neural probe (the "NeuroWeb") consisting of hexagonal boron nitride and graphene, which leverages the strengths of both surface electrode array and implantable multi-electrode array. The NeuroWeb open lattice structure with a total thickness of 100 nm demonstrates high flexibility and strong adhesion, establishing a conformal and tight interface with the uneven mouse brain surface. In vivo electrophysiological recordings show that NeuroWeb detects stable single-unit activity of neurons with high signal-to-noise ratios. Furthermore, we investigate neural interactions between the somatosensory cortex and the cerebellum using transparent dual NeuroWebs and optical stimulation, and measure the times of neural signal transmission between the brain regions depending on the pathway. Therefore, NeuroWeb can be expected to pave the way for understanding complex brain networks with optical and electrophysiological mapping of the brain.

Tracing multiple scattering trajectories for deep optical imaging in scattering media.

Multiple light scattering hampers imaging objects in complex scattering media. Approaches used in real practices mainly aim to filter out multiple scattering obscuring the ballistic waves that travel straight through the scattering medium. Here, we propose a method that makes the deterministic use of multiple scattering for microscopic imaging of an object embedded deep within scattering media. The proposed method finds a stack of multiple complex phase plates that generate similar light trajectories as the original scattering medium. By implementing the inverse scattering using the identified phase plates, our method rectifies multiple scattering and amplifies ballistic waves by almost 600 times. This leads to a significant increase in imaging depth-more than three times the scattering mean free path-as well as the correction of image distortions. Our study marks an important milestone in solving the long-standing high-order inverse scattering problems.

Label-free adaptive optics single-molecule localization microscopy for whole zebrafish.

Specimen-induced aberration has been a major factor limiting the imaging depth of single-molecule localization microscopy (SMLM). Here, we report the application of label-free wavefront sensing adaptive optics to SMLM for deep-tissue super-resolution imaging. The proposed system measures complex tissue aberrations from intrinsic reflectance rather than fluorescence emission and physically corrects the wavefront distortion more than three-fold stronger than the previous limit. This enables us to resolve sub-diffraction morphologies of cilia and oligodendrocytes in whole zebrafish as well as dendritic spines in thick mouse brain tissues at the depth of up to 102 µm with localization number enhancement by up to 37 times and localization precision comparable to aberration-free samples. The proposed approach can expand the application range of SMLM to whole zebrafish that cause the loss of localization number owing to severe tissue aberrations.

Computational conjugate adaptive optics microscopy for longitudinal through-skull imaging of cortical myelin.

Myelination processes are closely related to higher brain functions such as learning and memory. While their longitudinal observation has been crucial to understanding myelin-related physiology and various brain disorders, skull opening or thinning has been required to secure clear optical access. Here we present a high-speed reflection matrix microscope using a light source with a wavelength of 1.3 µm to reduce tissue scattering and aberration. Furthermore, we develop a computational conjugate adaptive optics algorithm designed for the recorded reflection matrix to optimally compensate for the skull aberrations. These developments allow us to realize label-free longitudinal imaging of cortical myelin through an intact mouse skull. The myelination processes of the same mice were observed from 3 to 10 postnatal weeks to the depth of cortical layer 4 with a spatial resolution of 0.79 µm. Our system will expedite the investigations on the role of myelination in learning, memory, and brain disorders.

Flexible-type ultrathin holographic endoscope for microscopic imaging of unstained biological tissues.

Ultrathin lensless fibre endoscopes offer minimally invasive investigation, but they mostly operate as a rigid type due to the need for prior calibration of a fibre probe. Furthermore, most implementations work in fluorescence mode rather than label-free imaging mode, making them unsuitable for general medical diagnosis. Herein, we report a fully flexible ultrathin fibre endoscope taking 3D holographic images of unstained tissues with 0.85-µm spatial resolution. Using a bare fibre bundle as thin as 200-µm diameter, we design a lensless Fourier holographic imaging configuration to selectively detect weak reflections from biological tissues, a critical step for label-free endoscopic reflectance imaging. A unique algorithm is developed for calibration-free holographic image reconstruction, allowing us to image through a narrow and curved passage regardless of fibre bending. We demonstrate endoscopic reflectance imaging of unstained rat intestine tissues that are completely invisible to conventional endoscopes. The proposed endoscope will expedite a more accurate and earlier diagnosis than before with minimal complications.

Through-skull brain imaging in vivo at visible wavelengths via dimensionality reduction adaptive-optical microscopy.

Compensation of sample-induced optical aberrations is crucial for visualizing microscopic structures deep within biological tissues. However, strong multiple scattering poses a fundamental limitation for identifying and correcting the tissue-induced aberrations. Here, we introduce a label-free deep-tissue imaging technique termed dimensionality reduction adaptive-optical microscopy (DReAM) to selectively attenuate multiple scattering. We established a theoretical framework in which dimensionality reduction of a time-gated reflection matrix can attenuate uncorrelated multiple scattering while retaining a single-scattering signal with a strong wave correlation, irrespective of sample-induced aberrations. We performed mouse brain imaging in vivo through the intact skull with the probe beam at visible wavelengths. Despite the strong scattering and aberrations, DReAM offered a 17-fold enhancement of single scattering-to-multiple scattering ratio and provided high-contrast images of neural fibers in the brain cortex with the diffraction-limited spatial resolution of 412 nanometers and a 33-fold enhanced Strehl ratio.

High-throughput volumetric adaptive optical imaging using compressed time-reversal matrix.

Deep-tissue optical imaging suffers from the reduction of resolving power due to tissue-induced optical aberrations and multiple scattering noise. Reflection matrix approaches recording the maps of backscattered waves for all the possible orthogonal input channels have provided formidable solutions for removing severe aberrations and recovering the ideal diffraction-limited spatial resolution without relying on fluorescence labeling and guide stars. However, measuring the full input-output response of the tissue specimen is time-consuming, making the real-time image acquisition difficult. Here, we present the use of a time-reversal matrix, instead of the reflection matrix, for fast high-resolution volumetric imaging of a mouse brain. The time-reversal matrix reduces two-way problem to one-way problem, which effectively relieves the requirement for the coverage of input channels. Using a newly developed aberration correction algorithm designed for the time-reversal matrix, we demonstrated the correction of complex aberrations using as small as 2% of the complete basis while maintaining the image reconstruction fidelity comparable to the fully sampled reflection matrix. Due to nearly 100-fold reduction in the matrix recording time, we could achieve real-time aberration-correction imaging for a field of view of 40 × 40 µm2 (176 × 176 pixels) at a frame rate of 80 Hz. Furthermore, we demonstrated high-throughput volumetric adaptive optical imaging of a mouse brain by recording a volume of 128 × 128 × 125 µm3 (568 × 568 × 125 voxels) in 3.58 s, correcting tissue aberrations at each and every 1 µm depth section, and visualizing myelinated axons with a lateral resolution of 0.45 µm and an axial resolution of 2 µm.

Laser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull.

A mouse skull is a barrier for high-resolution optical imaging because its thick and inhomogeneous internal structures induce complex aberrations varying drastically from position to position. Invasive procedures creating either thinned-skull or open-skull windows are often required for the microscopic imaging of brain tissues underneath. Here, we propose a label-free imaging modality termed laser scanning reflection-matrix microscopy for recording the amplitude and phase maps of reflected waves at non-confocal points as well as confocal points. The proposed method enables us to find and computationally correct up to 10,000 angular modes of aberrations varying at every 10 × 10 µm2 patch in the sample plane. We realized reflectance imaging of myelinated axons in vivo underneath an intact mouse skull, with an ideal diffraction-limited spatial resolution of 450 nm. Furthermore, we demonstrated through-skull two-photon fluorescence imaging of neuronal dendrites and their spines by physically correcting the aberrations identified from the reflection matrix.

Deep tissue space-gated microscopy via acousto-optic interaction.

To extend the imaging depth of high-resolution optical microscopy, various gating operations-confocal, coherence, and polarization gating-have been devised to filter out the multiply scattered wave. However, the imaging depth is still limited by the multiply scattered wave that bypasses the existing gating operations. Here, we present a space gating method, whose mechanism is independent of the existing methods and yet effective enough to complement them. Specifically, we reconstruct an image only using the ballistic wave that is acousto-optically modulated at the object plane. The space gating suppresses the multiply scattered wave by 10-100 times in a highly scattering medium, and thus enables visualization of the skeletal muscle fibers in whole-body zebrafish at 30 days post fertilization. The space gating will be an important addition to optical-resolution microscopy for achieving the ultimate imaging depth set by the detection limit of ballistic wave.

Label-free neuroimaging in vivo using synchronous angular scanning microscopy with single-scattering accumulation algorithm.

Label-free in vivo imaging is crucial for elucidating the underlying mechanisms of many important biological systems in their most native states. However, the applicability of existing modalities has been limited to either superficial layers or early developmental stages due to tissue turbidity. Here, we report a synchronous angular scanning microscope for the rapid interferometric recording of the time-gated reflection matrix, which is a unique matrix characterizing full light-specimen interaction. By applying single scattering accumulation algorithm to the recorded matrix, we removed both high-order sample-induced aberrations and multiple scattering noise with the effective aberration correction speed of 10,000 modes/s. We demonstrated in vivo imaging of whole neural network throughout the hindbrain of the larval zebrafish at a matured stage where physical dissection used to be required for conventional imaging. Our method will expand the scope of applications for optical imaging, where fully non-invasive interrogation of living specimens is critical.

Association Between Vitamin D Insufficiency and Metabolic Syndrome in Patients With Psychotic Disorders.

OBJECTIVE: This study examined the association between vitamin D and metabolic syndrome in patients with psychotic disorders. METHODS: The study enrolled 302 community-dwelling patients with psychotic disorders. Sociodemographic and clinical characteristics, including blood pressure, physical activity, and dietary habit were gathered. Laboratory examinations included vitamin D, lipid profile, fasting plasma glucose, HbA1c, liver function, and renal function. Vitamin D insufficiency was defined as <20 ng/mL. Clinical characteristics associated with vitamin D insufficiency were identified. RESULTS: Among the 302 participants, 236 patients (78.1%) had a vitamin D insufficiency and 97 (32.1%) had metabolic syndrome. Vitamin D insufficiency was significantly associated with the presence of metabolic syndrome (p=0.006) and hypertension (p=0.017). Significant increases in triglycerides and alanine transaminase were observed in the group with a vitamin D insufficiency (p=0.002 and 0.011, respectively). After adjusting for physical activity and dietary habit scores, vitamin D insufficiency remained significantly associated with metabolic syndrome and hypertension. CONCLUSION: Vitamin D insufficiency was associated with metabolic syndrome and was particularly associated with high blood pressure, although the nature, direction and implications of this association are unclear.

High-resolution adaptive optical imaging within thick scattering media using closed-loop accumulation of single scattering.

Thick biological tissues give rise to not only the multiple scattering of incoming light waves, but also the aberrations of remaining signal waves. The challenge for existing optical microscopy methods to overcome both problems simultaneously has limited sub-micron spatial resolution imaging to shallow depths. Here we present an optical coherence imaging method that can identify aberrations of waves incident to and reflected from the samples separately, and eliminate such aberrations even in the presence of multiple light scattering. The proposed method records the time-gated complex-field maps of backscattered waves over various illumination channels, and performs a closed-loop optimization of signal waves for both forward and phase-conjugation processes. We demonstrated the enhancement of the Strehl ratio by more than 500 times, an order of magnitude or more improvement over conventional adaptive optics, and achieved a spatial resolution of 600 nm up to an imaging depth of seven scattering mean free paths.

Multi-stability of circadian phase wave within early postnatal suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is a group of cells that functions as a biological master clock. In different SCN cells, oscillations of biochemical markers such as the expression-level of clock genes, are not synchronized but instead form slow circadian phase waves propagating over the whole cell population spatio-temporal structure is a fixed property set by the anatomy of a given SCN. Here, we show that this is not the case in early postnatal SCN. Earlier studies presumed that their Based on bioluminescence imaging experiments with Per2-Luciferase mice SCN cultures which guided computer simulations of a realistic model of the SCN, we demonstrate that the wave is not unique but can be in various modes including phase- coherent oscillation, crescent-shaped wave, and most notably, a rotating pinwheel wave that conceptually resembles a wall clock with a rotating hand. Furthermore, mode transitions can be induced by a pulse of 38.5 °C temperature perturbation. Importantly, the waves support a significantly different period, suggesting that neither a spatially-fixed phase ordering nor a specialized pacemaker having a fixed period exist in these studied SCNs. These results lead to new important questions of what the observed multi-stability means for the proper function of an SCN and its arrhythmia.

Serotonin-2C receptor involved serotonin-induced Ca²âº mobilisations in neuronal progenitors and neurons in rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals, undergoes serotonergic regulation, but the underlying mechanisms remain obscure. Here, we generated a subclone of an SCN progenitor cell line expressing Ca(2+) sensors (SCN2.2YC) and compared its 5-HT receptor signalling with that of rat SCN neurons in brain slices. SCN2.2YC cells expressed 5-HT1A/2A/2B/2C, but not 5A/7, while all six subtypes were expressed in SCN tissues. High K(+) or 5-HT increased cytosolic Ca(2+) in SCN2.2YC cells. The 5-HT responses were inhibited by ritanserin and SB-221284, but resistant to WAY-100635 and RS-127445, suggesting predominant involvement of 5-HT2C for Ca(2+) mobilisations. Consistently, Ca(2+) imaging and voltage-clamp electrophysiology using rat SCN slices demonstrated post-synaptic 5-HT2C expression. Because 5-HT2C expression was postnatally increased in the SCN and 5-HT-induced Ca(2+) mobilisations were amplified in differentiated SCN2.2YC cells and developed SCN neurons, we suggest that this signalling development occurs in accordance with central clock maturations.

Circadian waves of cytosolic calcium concentration and long-range network connections in rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master clock in mammals governing the daily physiological and behavioral rhythms. It is composed of thousands of clock cells with their own intrinsic periods varying over a wide range (20-28 h). Despite this heterogeneity, an intact SCN maintains a coherent 24 h periodic rhythm through some cell-to-cell coupling mechanisms. This study examined how the clock cells are connected to each other and how their phases are organized in space by monitoring the cytosolic free calcium ion concentration ([Ca(2+)](c)) of clock cells using the calcium-binding fluorescent protein, cameleon. Extensive analysis of 18 different organotypic slice cultures of the SCN showed that the SCN calcium dynamics is coordinated by phase-synchronizing networks of long-range neurites as well as by diffusively propagating phase waves. The networks appear quite extensive and far-reaching, and the clock cells connected by them exhibit heterogeneous responses in their amplitudes and periods of oscillation to tetrodotoxin treatments. Taken together, our study suggests that the network of long-range cellular connectivity has an important role for the SCN in achieving its phase and period coherence.

Inactivation of glycogen synthase kinase-3ß is required for osteoclast differentiation.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3ß in osteoblasts is well characterized as a negative regulator of ß-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3ß inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3ß activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3ß (GSK3ß-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3ß (GSK3ß-K85R) or small interfering RNA (siRNA)-mediated GSK-3ß silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3ß further confirmed the negative role of GSK-3ß in osteoclast formation. We also show that overexpression of the GSK3ß-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3ß-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3ß in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.

[A case of tuberculous peritonitis developed during chemotherapy for tuberculous pleurisy as paradoxical response].

After the start of anti-tuberculous treatment, paradoxical worsening of tuberculous lesions has been described. However, abdominal tuberculosis as paradoxical response is relatively rare. This report describes the 26-year-old female who suffered from peritoneal tuberculosis while treating tuberculous pleurisy with anti-tuberculous medications. It was considered as paradoxical response, rather than treatment failure or else. She was successfully managed with continuing initial anti-tuberculous medications. When a patient on anti-tuberculous medications is presented with abdominal symptoms, the possibility of paradoxical response should be considered to avoid unnecessary tests and treatments, which may result in more suffering of the patient. Herein, we report a case of peritoneal tuberculosis as paradoxical response while treating tuberculous pleurisy.

Intracellular calcium spikes in rat suprachiasmatic nucleus neurons induced by BAPTA-based calcium dyes.

BACKGROUND: Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of "Ca(2+) spikes" (i.e., [Ca(2+)](c) transients having a bandwidth of 10 approximately 100 seconds) in SCN neurons, but it is unclear if these SCN Ca(2+) spikes are related to the slow circadian rhythms. METHODOLOGY/PRINCIPAL FINDINGS: We addressed this issue based on a Ca(2+) indicator dye (fluo-4) and a protein Ca(2+) sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca(2+) spikes in 18% of rat SCN cells in acute brain slices, but the Ca(2+) spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca(2+) spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca(2+) spikes was increased to 13 approximately 14%. CONCLUSIONS/SIGNIFICANCE: Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca(2+) spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca(2+) spiking activity is caused by the Ca(2+) chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca(2+)](c) in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca(2+) spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca(2+) spikes in the function of SCN.

[A case of spontaneous regression of hepatocellular carcinoma with multiple lung metastases].

Spontaneous regression of hepatocellular carcinoma (HCC) is extremely rare. We report a case of 67-year-old man having HBV-associated HCC with multiple lung metastases which regressed spontaneously. The patient had single liver mass and received surgical resection. The mass was confirmed as HCC histopathologically. Nine years after surgical resection, a 3.3 cm sized recurred HCC was detected on the resection margin in CT scan. Transarterial chemoembolization (TACE) was performed 3 times, and lung metastases developed thereafter. The patient received 2 more sessions of TACE, however, metastatic lung nodules were in progress very rapidly. We decided to stop TACE and followed the patient regularly without any anti-cancer treatment. Nine months after development of lung metastasis, the size and number of metastatic lung nodules decreased and were not detected anymore after 14 months. Serum alpha-fetoprotein levels also decreased to normal range and no viable tumor was noted in the liver. The patient is still alive 12 years after the first diagnosis of HCC and 16 months after lung metastasis developed.