Brain representations of affective valence and intensity in sustained pleasure and pain.

Pleasure and pain are two fundamental, intertwined aspects of human emotions. Pleasurable sensations can reduce subjective feelings of pain and vice versa, and we often perceive the termination of pain as pleasant and the absence of pleasure as unpleasant. This implies the existence of brain systems that integrate them into modality-general representations of affective experiences. Here, we examined representations of affective valence and intensity in an functional MRI (fMRI) study (n = 58) of sustained pleasure and pain. We found that the distinct subpopulations of voxels within the ventromedial and lateral prefrontal cortices, the orbitofrontal cortex, the anterior insula, and the amygdala were involved in decoding affective valence versus intensity. Affective valence and intensity predictive models showed significant decoding performance in an independent test dataset (n = 62). These models were differentially connected to distinct large-scale brain networks-the intensity model to the ventral attention network and the valence model to the limbic and default mode networks. Overall, this study identified the brain representations of affective valence and intensity across pleasure and pain, promoting a systems-level understanding of human affective experiences.

Statistically unbiased prediction enables accurate denoising of voltage imaging data.

Here we report SUPPORT (statistically unbiased prediction utilizing spatiotemporal information in imaging data), a self-supervised learning method for removing Poisson-Gaussian noise in voltage imaging data. SUPPORT is based on the insight that a pixel value in voltage imaging data is highly dependent on its spatiotemporal neighboring pixels, even when its temporally adjacent frames alone do not provide useful information for statistical prediction. Such dependency is captured and used by a convolutional neural network with a spatiotemporal blind spot to accurately denoise voltage imaging data in which the existence of the action potential in a time frame cannot be inferred by the information in other frames. Through simulations and experiments, we show that SUPPORT enables precise denoising of voltage imaging data and other types of microscopy image while preserving the underlying dynamics within the scene.

Thermally Managed, Injectable Optoelectronic Probe with Heat Dissipation Guide for Photodynamic Therapy.

The development of fabrication technologies and appearance of new materials has resulted in dramatic increase in the performance of electronic devices, while the overall size has decreased. Recent electronic devices made of micro/nano-size components show high efficiency and outstanding performance with compact size, but these devices have revealed several fatal problems. In particular, the isolated heat that is generated by numerous components concentrated in a limited small area at high density, such as bio-integrated devices, is an issue that needs to be urgently addressed, because it is closely related to the performance and lifetime of electronic devices. To solve these problems, the microscale light emitting diode (µLED)-based neural probe is introduced on an injectable heat dissipation guide. The heat dissipation guide is made of boron nitride (BN) nanomaterials with high thermal conductivity. The heat management noticeably improves the optical output performance of the µLEDs, in which BN effectively dissipates heat, and allows enhanced lighting from the LEDs to be transmitted through brain tissue without thermal damage. Moreover, it shows remarkable improvement in the therapeutic effect of photodynamic therapy of mouse cancer cells.

Honeycomb Artifact Removal Using Convolutional Neural Network for Fiber Bundle Imaging.

We present a new deep learning framework for removing honeycomb artifacts yielded by optical path blocking of cladding layers in fiber bundle imaging. The proposed framework, HAR-CNN, provides an end-to-end mapping from a raw fiber bundle image to an artifact-free image via a convolution neural network (CNN). The synthesis of honeycomb patterns on ordinary images allows conveniently learning and validating the network without the enormous ground truth collection by extra hardware setups. As a result, HAR-CNN shows significant performance improvement in honeycomb pattern removal and also detailed preservation for the 1961 USAF chart sample, compared with other conventional methods. Finally, HAR-CNN is GPU-accelerated for real-time processing and enhanced image mosaicking performance.

Multiplexed expansion microscopy of the brain through fluorophore screening.

Super-resolution microscopy techniques have been widely adopted in biological sciences. Recently, a new super-resolution microscopy technique, called expansion microscopy (ExM) has been developed. In this technique, biomolecules inside specimens are first labeled with fluorophores, followed by in-situ hydrogel synthesis and physical expansion of the specimens. Image quality, including brightness and signal-to-noise ratio, depends on the extent to which fluorophores have bleached during the in-situ hydrogel synthesis process. In this work, we compared the fluorescence signal brightness of more than 20 fluorophores, after expansion, to identify the best fluorophore set for 4-color expansion microscopy imaging. In addition, we achieved 5-color multiplexed expansion microscopy by photo-bleaching one of the four fluorophores and re-staining thereafter.

Spectral Reflectometry in Biomedical Imaging and Sensing.

Spectral reflectometry is a spectroscopic measurement technique based on thin-film interference, which has been widely applied in industries to measure thicknesses of thin dielectric layers at the nanoscale. Recent advances in the understanding of biological nanostructures have opened a new field of spectral reflectometry in biomedicine from molecular level sensing to biomedical imaging. This chapter comprehensively covers the relevant topics on spectral reflectometry in biomedicine from its principle to applications.

De Novo Shoot Regeneration Controlled by HEN1 and TCP3/4 in Arabidopsis.

Plants have the ability to regenerate whole plant body parts, including shoots and roots, in vitro from callus derived from a variety of tissues. However, the underlying mechanisms for this de novo organogenesis, which is based on the totipotency of callus cells, are poorly understood. Here, we report that a microRNA (miRNA)-mediated posttranscriptional regulation plays an important role in de novo shoot regeneration. We found that mutations in HUA ENHANCER 1 (HEN1), a gene encoding a small RNA methyltransferase, cause cytokinin-related defects in de novo shoot regeneration. A hen1 mutation caused a large reduction in the miRNA319 (miR319) level and a subsequent increase in its known target (TCP3 and TCP4) transcript levels. TCP transcription factors redundantly inhibited shoot regeneration and directly activated the expression of a negative regulator of cytokinin response ARABIDOPSIS THALIANA RESPONSE REGULATOR 16 (ARR16). A tcp4 mutation at least partly rescued the shoot-regeneration defect and derepression of ARR16 in hen1. These findings demonstrate that the miR319-TCP3/4-ARR16 axis controls de novo shoot regeneration by modulating cytokinin responses.

Improving collection efficiency in two-photon endoscopy with reflective waveguiding.

Two-photon endoscopy based on a gradient-index lens has been widely utilized for studying cellular behaviors in deep-lying tissues with minimal invasiveness in vivo. Although the efficient collection of emitted light is critical to attain high-contrast spatiotemporal information, the intrinsic low numerical aperture of the endoscopic probe poses a physical limitation. We report a simple solution to overcome this limit by incorporating a reflective waveguide ensheathing the endoscopic probe, which improves the collection efficiency by approximately two-fold. We describe its principle, fabrication procedure, optical characterization, and utilities in biological tissues.

In vivo fluorescence microscopy: lessons from observing cell behavior in their native environment.

Microscopic imaging techniques to visualize cellular behaviors in their natural environment play a pivotal role in biomedical research. Here, we review how recent technical advances in intravital microscopy have enabled unprecedented access to cellular physiology in various organs of mice in normal and diseased states.

Bioorthogonal Click Chemistry-Based Synthetic Cell Glue.

Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans-cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm(-2) . Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell-cell interactions in vivo.

Card9 mediates intestinal epithelial cell restitution, T-helper 17 responses, and control of bacterial infection in mice.

BACKGROUND & AIMS: Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection. METHODS: We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis. RESULTS: Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria. CONCLUSIONS: The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice.

In vivo imaging of Lgr5-positive cell populations using confocal laser endomicroscopy during early colon tumorigenesis.

BACKGROUND AND STUDY AIMS: A diagnostic molecular marker for pre-neoplastic lesions, particularly before polyposis, is still lacking. Lgr5 has been broadly accepted as a marker for intestinal cancer stem cells. The aim of this study was to investigate the monitoring of Lgr5( + ) cells as a useful tool for the early diagnosis of premalignant lesions before polyp formation. METHODS: In vivo molecular imaging was performed to examine colon tumorigenesis in Lgr5-EGFP mice treated with azoxymethane and dextran sodium sulfate. eGFP( +) Lgr5( +) regions in the descending colon were longitudinally monitored using side-view confocal endomicroscopy. Based on the eGFP signal intensity on the luminal surface, polyps were classified into two groups - Lgr5-high and Lgr5-low. White light colonoscopy was used to monitor polyp formation. RESULTS: Approximately 75 % of the polyps originated from foci containing Lgr5-eGFP( +) cells, whereas 25 % of the polyps emerged from Lgr5( -) foci. Among eGFP( +) foci, Lgr5-high foci grew faster than Lgr5-low foci. CONCLUSIONS: Polyps developed at Lgr5( +) regions. Luminal Lgr5 expression was correlated with the growth rate of early-stage adenomas. Lgr5 is a promising molecular marker for the early diagnosis of colon tumors.

Minimally invasive molecular delivery into the brain using optical modulation of vascular permeability.

Systemic delivery of bioactive molecules in the CNS is hampered by the blood-brain barrier, which has bottlenecked noninvasive physiological study of the brain and the development of CNS drugs. Here we report that irradiation with an ultrashort pulsed laser to the blood vessel wall induces transient leakage of blood plasma without compromising vascular integrity. By combining this method with a systemic injection, we delivered target molecules in various tissues, including the brain cortex. This tool allows minimally invasive local delivery of chemical probes, nanoparticles, and viral vectors into the brain cortex. Furthermore, we demonstrated astrocyte-mediated vasodilation in vivo without opening the skull, using this method to load a calcium indicator in conjunction with label-free photoactivation of astrocytes.

In vivo femtosecond endosurgery: an intestinal epithelial regeneration-after-injury model.

Regeneration of the intestinal epithelium after injury or during pathogenesis is a dynamic cellular process critical for host immunity. However, current epithelial injury models provide poor spatial control, complicating the study of precise cellular responses. Here we developed endoscopic femtosecond-laser surgery capable of generating acute tissue injury. A side-view probe provides a convenient access to the distal colon in the mouse in vivo and allows real-time intraoperative monitoring as well as pre- and post-surgery examinations via multiphoton imaging. The photo-induced damage showed a nonlinear dependence on laser intensity. At an optical power of 200 mW (2.5 nJ per pulse), scanning the beam focus over 300x300 µm(2) area in the colonic mucosa generated substantial vascular damages within 30 s. We confirmed the localized tissue damage and the physiologic regeneration of the disrupted epithelium by in situ barrier function assays, validating the animal model for epithelial regeneration following injury. The femtosecond endosurgery technique is applicable to various experimental models based on laser-induced perturbations.

All-optical observation on activity-dependent nanoscale dynamics of myelinated axons.

Significance: In the mammalian brain, rapid conduction of neural information is supported by the myelin, the functional efficacy of which shows steep dependence on its nanoscale cytoarchitecture. Although previous in vitro studies have suggested that neural activity accompanies nanometer-scale cellular deformations, whether neural activity can dynamically remodel the myelinated axon has remained unexplored due to the technical challenge in observing its nanostructural dynamics in living tissues. Aim: We aim to observe activity-dependent nanostructural dynamics of myelinated axons in a living brain tissue. Approach: We introduced a novel all-optical approach combining a nanoscale dynamic readout based on spectral interferometry and optogenetic control of neural excitation in an acute brain slice preparation. Results: In response to optogenetically evoked neuronal burst firing, the myelinated axons exhibited progressive and reversible spectral redshifts, corresponding to the transient swelling at a subnanometer scale. We further revealed that the activity-dependent nanostructural dynamics was localized to the paranode. Conclusions: Our all-optical studies substantiate that myelinated axon exhibits activity-dependent nanoscale swelling, which potentially serves to dynamically tune the transmission speed of neural information.

Whole-brain mapping of effective connectivity by fMRI with cortex-wide patterned optogenetics.

Functional magnetic resonance imaging (fMRI) with optogenetic neural manipulation is a powerful tool that enables brain-wide mapping of effective functional networks. To achieve flexible manipulation of neural excitation throughout the mouse cortex, we incorporated spatiotemporal programmable optogenetic stimuli generated by a digital micromirror device into an MRI scanner via an optical fiber bundle. This approach offered versatility in space and time in planning the photostimulation pattern, combined with in situ optical imaging and cell-type-specific or circuit-specific genetic targeting in individual mice. Brain-wide effective connectivity obtained by fMRI with optogenetic stimulation of atlas-based cortical regions is generally congruent with anatomically defined axonal tracing data but is affected by the types of anesthetics that act selectively on specific connections. fMRI combined with flexible optogenetics opens a new path to investigate dynamic changes in functional brain states in the same animal through high-throughput brain-wide effective connectivity mapping.

Implantable acousto-optic window for monitoring ultrasound-mediated neuromodulation in vivo.

Significance: Ultrasound has recently received considerable attention in neuroscience because it provides noninvasive control of deep brain activity. Although the feasibility of ultrasound stimulation has been reported in preclinical and clinical settings, its mechanistic understanding remains limited. While optical microscopy has become the "gold standard" tool for investigating population-level neural functions in vivo, its application for ultrasound neuromodulation has been technically challenging, as most conventional ultrasonic transducers are not designed to be compatible with optical microscopy. Aim: We aimed to develop a transparent acoustic transducer based on a glass coverslip called the acousto-optic window (AOW), which simultaneously provides ultrasound neuromodulation and microscopic monitoring of neural responses in vivo. Approach: The AOW was fabricated by the serial deposition of transparent acoustic stacks on a circular glass coverslip, comprising a piezoelectric material, polyvinylidene fluoride-trifluoroethylene, and indium-tin-oxide electrodes. The fabricated AOW was implanted into a transgenic neural-activity reporter mouse after open craniotomy. Two-photon microscopy was used to observe neuronal activity in response to ultrasonic stimulation through the AOW. Results: The AOW allowed microscopic imaging of calcium activity in cortical neurons in response to ultrasound stimulation. The optical transparency was ∼ 40 % over the visible and near-infrared spectra, and the ultrasonic pressure was 0.035 MPa at 10 MHz corresponding to 10 mW / cm 2 . In anesthetized Gad2-GCaMP6-tdTomato mice, we observed robust ultrasound-evoked activation of inhibitory cortical neurons at depths up to 200 µ m . Conclusions: The AOW is an implantable ultrasonic transducer that is broadly compatible with optical imaging modalities. The AOW will facilitate our understanding of ultrasound neuromodulation in vivo.

Advances in Optical Tools to Study Taste Sensation.

Taste sensation is the process of converting chemical identities in food into a neural code of the brain. Taste information is initially formed in the taste buds on the tongue, travels through the afferent gustatory nerves to the sensory ganglion neurons, and finally reaches the multiple taste centers of the brain. In the taste field, optical tools to observe cellular-level functions play a pivotal role in understanding how taste information is processed along a pathway. In this review, we introduce recent advances in the optical tools used to study the taste transduction pathways.

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.

Blockade of vascular endothelial growth factor sensitizes tumor-associated vasculatures to angiolytic therapy with a high-frequency ultrashort pulsed laser.

Because of high spatial resolution and superior tissue penetration, a femtosecond laser of the near-infrared spectrum has great potential to improve the efficacy of conventional photodynamic therapy; however, the lack of suitable photosensitizers has so far limited its bedside applications. Recently, our group reported that a brief irradiation by femtosecond lasers in the absence of exogenous probes can modulate various cellular behaviors in vitro and in vivo. Here, we demonstrate that targeted irradiation by a femtosecond laser disrupted tumor-associated blood vessels, and the inhibition of vascular endothelial growth factor signaling augmented the efficacy of laser-induced angiolysis. Further, we show that reactive oxygen species (ROS) are generated in response to laser irradiation, and reducing the intracellular levels of ROS rendered endothelial cells resistant to laser-induced cytotoxicity. Collectively, these results indicate that a femtosecond laser can be used as a vascular-disrupting therapeutic modality for cancer treatment, especially when used in combination with conventional anti-angiogenic therapies.