High-throughput screening techniques are pivotal to unlocking the mysteries of biology. Yet, the promise of droplet microfluidics in enabling single-cell resolution, ultra-high-throughput screening remains largely unfulfilled. Droplet sorting errors caused by polydisperse droplet sizes that are often inevitable in multi-step assays have severely limited the effectiveness and utility of this technique, especially when screening large libraries. Even a relatively low 1% sorting error results in 10,000 false calls in a 1,000,000 droplet screen, imposing an unreasonably large burden on downstream validation. Here, we present NOVAsort (Next-generation Opto-Volume-based Accurate droplet sorter), a device capable of discerning droplets based on both size and fluorescence intensity. With a 1000- and 10,000-fold reduction in false positives and false negatives, respectively. NOVAsort addresses the challenges of conventional droplet sorting approaches and sets standards for accuracy and throughput in droplet microfluidic assays.

Infrared thermal imaging has been widely used to show the correlation between thermal characteristics of the body and muscle activation. This study aims to investigate a method using thermal imaging to visualize and differentiate target muscles during resistance training. Thermal images were acquired to monitor three target muscles (i.e., biceps brachii, triceps brachii, and deltoid muscle) in the brachium while varying the training weight, duration, and order of training. The acquired thermal images were segmented and converted to heat maps. By generating difference heat maps from pairs of heat maps during training, the target muscles were clearly visualized, with an average temperature difference of 0.86 °C. It was observed that training order had no significant effect on skin surface temperature. The difference heat maps were also used to train a convolutional neural network (CNN) to show the feasibility of target muscle classification, with an accuracy of 92.3%. This study demonstrated that infrared thermal imaging could be effectively utilized to locate and differentiate target muscle activation during resistance training.

Optogenetics has the potential to transform the study of the peripheral nervous system (PNS), but the complex anatomy of the PNS poses unique challenges for the focused delivery of light to specific tissues. This protocol describes the fabrication of a wireless telemetry system for studying peripheral sensory pathways. Unlike existing wireless approaches, the low-power wireless telemetry offers organ specificity via a sandwiched pre-curved tether, and enables high-throughput analysis of behavioral experiments with a channel isolation strategy. We describe the technical procedures for the construction of these devices, the wireless power transmission (TX) system with antenna coils, and their implementation for in vivo experimental applications. In total, the timeline of the procedure, including device fabrication, implantation, and preparation to begin in vivo experimentation can be completed in ~2-4 weeks. Implementation of these devices allows for chronic (>1 month) wireless optogenetic manipulation of peripheral neural pathways in freely behaving animals navigating homecage environments (up to 8)., [摘要]光遗传学有可能改变周围神经系统 (PNS) 的研究，但 PNS 的复杂解剖结构对将光集中传递到特定组织提出了独特的挑战。该协议描述了用于研究外围感觉通路的无线遥测系统的制造。与现有的无线方法不同，低功率无线遥测通过夹在中间的预弯曲系绳提供器官特异性，并通过通道隔离策略对行为实验进行高通量分析。我们描述了构建这些设备的技术程序、带有天线线圈的无线电力传输 (TX) 系统，以及它们在体内实验应用中的实现。总的来说，该程序的时间表，包括设备制造、植入和准备开始体内实验，可以在 ~ 2-4 周内完成。这些设备的实施允许在自由行为的动物（最多 8 个）中对周围神经通路进行慢性（>1 个月）无线光遗传学操作。[背景]光遗传学通过对基因定义的神经细胞类型进行有针对性的操作，改变了神经科学（ Deisseroth ，2011 和 2015）。这通常涉及使用 Cre 驱动鼠标表达光激活离子通道并将光纤植入大脑（Park等人，2015b、2015a 和 2016）。通过这种方式，可以用光激活或灭活特定细胞类型，从而测试它们在行为中的因果作用（Kim等人，2013；Montgomery等人，2015；Shin等人，2017）。光遗传学也有可能改变 PNS 的研究，但 PNS 的复杂解剖结构对将光集中传递到特定组织提出了独特的挑战（Towne等人，2013；Montgomery等人，2016； Maimon 等人，2018）。 PNS 由感觉和运动神经元组成，细胞体分布在广泛分布的外周神经节中。主要的感觉神经节包括背根神经节，它由沿脊柱长度两侧排列的脊髓感觉神经元的细胞体簇组成，以及包含迷走神经感觉神经元细胞体的结节神经节（Berthoud 和Neuhuber ，2000 ）。这两种感觉系统广泛地支配着一系列在大小、形状、位置和可及性方面不同的外周器官，因此需要个性化的策略来将光传递到不同的组织（Berthoud，2008；de Lartigue ，2016；Williams等人，2016） .此外，这些器官中的一些在正常生理或行为期间是高度可移动的（例如，在消化期间肠的运动），这进一步使光纤的定位和锚定复杂化。因此，与大脑不同，开发稳健的策略以选择性地将光传递到自由行为动物的外周组织是一项相当大的挑战。Caggiano ）用光靶向外周组织 等人，2016； Hibberd等人，2018 年），类似于大脑中常用的颅内光纤。虽然这种方法的优点是它可以很容易地与现成的组件（例如，激光和 LED）耦合，但腹部内脏与大脑不同，因为它缺乏用于固定大型光纤和防止运动。此外，大多数光纤的不灵活特性会在正常动物运动期间导致组织和神经或光纤本身的剪切。出于这个原因，有线光纤很少用于行为动物的外围光遗传学（ Maimon 等人，2017 年和 2018 年； Towne等人，2013 年）。另一种方法是植入一个无线探头，将光源内部化，从而绕过与光纤电缆相关的物理限制。最近已经描述了这种无线、射频( RF) 供电设备的策略（Montgomery等人，2015 和 2016 年；Park等人，2015a 和 2015b），但实现长期、器官限制照明，不妨碍正常行为或生理。例如，无线供电的微型发光二极管 (µLED) 已使用环形弹性体套管固定在大鼠膀胱上（Mickle等人，2019 年），但该装置阻碍了正常的器官扩张。已经报道了一种将 µLED 缝合到心脏表面的无线柔性设备（ Gutruf et al ., 2019)，但它的功能没有被证明超过八天，排除了大多数行为研究。更一般地说，将任何 µLED 固定到目标器官表面会导致光反向散射和附近组织的非特异性光遗传学照明。最近，我们开发并发布了一类新的可植入无线设备，展示了它们在研究迷走神经方面的适用性（Kim等人，2021 年）。这种新的无线光遗传学植入物通过预先弯曲的系绳从胃内部向神经末梢传递光。这种胃内光传输能够照亮胃支配神经，比贴在器官表面的 µLED 具有更高的特异性，并且原则上可以扩展到研究其他器官，例如肠道。在这里，我们描述了制造这些设备及其体内应用的技术程序。

Droplet microfluidics-based high-throughput screening (HTS) has proven to be an effective method for rapidly analyzing broad ranges of biological samples, such as cell libraries. However, the final step of the droplet microfluidics-based screening workflow, which is to dispense the sorted “hit” droplets to off-chip, ideally one “hit” droplet at a time, is prone to high error, especially when droplet size variation is present. Such size variations, often unavoidable in complex multi-step droplet microfluidics assay, lead to variations in droplet transition speed as they flow towards the dispensing tip, where smaller droplets move faster than larger droplets within a microchannel. Such difference in transit speed causes simultaneous dispensing of multiple droplets at a time instead of single-droplet dispensing, resulting in “hit” cells in a dispensed droplet being mixed with those from another “hit” droplet. To address this challenge, an approach that uses blank spacing droplets as physical barriers between “hit” droplets so that “hit” droplet-to-droplet distance remains the same throughout the droplet transition process is proposed here. These blank droplets (up to 1000) and a single “hit” droplet flow out of the droplet dispensing tip, forming a “drip”, which is then dispensed one “drip” at a time into a well of a well plate or on an agar plate with sufficient drip-to-drip distance. This method of mixing empty droplets with “hit” droplets, combined with an off-the-shelf distance sensor that detects the formation of a “drip”, which then moves the dispensing plate upward using a linear motor for the formed “drip” to be dispensed, enables precise and automated single-drip (i.e., one “hit” droplet) dispensing even with polydisperse droplets. The developed system demonstrated a droplet dispensing accuracy of 99.9%, with a throughput of up to 8640 single drips (i.e., single “hit” droplets) per hour. The system's effectiveness was demonstrated through a droplet microfluidics-based antimicrobial susceptibility test (AST) assay, where four resistant strains from a mixture of 11 strains could be successfully identified. By smoothly transitioning droplet-encapsulated samples of interest identified through high-throughput droplet microfluidics assays to traditional biological assay workflow, this system offers a highly efficient, accurate, and cost-effective solution for conducting complex droplet microfluidics-based assays followed by further off-chip assays.

Microphysiological systems (MPS) have shown their capabilities in mimicking in vivo-like structural and functional complexity and are seeing significant increase in their utilization in the field of drug discovery and toxicology. However, the major time-consuming steps in the fabrication, utilization, and analyses of MPS devices limit the throughput for broader adoption. Here, we advanced the previously developed two-chamber MPS model of the female reproductive tracts from a single unit chip to an array type chip that is compatible with multi-channel pipettor or automated liquid handling robot for rapid and more efficient operation. To enable this array model, a new microfabrication method was developed, incorporating a microplate holder, bonding guide plate, and soft lithography cassette to minimize device-to-device variation. To validate its compatibility with multi-channel pipettors in chemical toxicity testing, cadmium, a chemical previously shown to elicit cytotoxicity in the two-chamber feto-maternal interface MPS model, was utilized to demonstrate highly uniform cell loading (variance < 100 cells/mm ² ) and consistent dose-dependent cytotoxic response. Additionally, a liquid handling robotic system was also utilized, with no operational errors such as air bubble introduction (zero bubbles out of 100 devices) during cell/chemical loading process, and no unintended cytotoxic effects (> 97% viability). These results highlight that this automation-compatible array type MPS device can provide highly consistent cell culture performance and significantly reduced chip-to-chip and operation-to-operation variations, overcoming the limitations of typical MPS devices.

Image sensors can achieve non‐contact detection of heart rate to predict the physiological status of the driver in an automotive driver monitoring system. However, the performance of such methods depends on the intensity of the light source. In this study, the effects of visible (VIS) and near‐infrared (NIR) light sources on heart rate measurement are investigated. The custom‐built setup employs Complementary Metal Oxide Semiconductor (CMOS) image sensors for visible and dual visible and near‐infrared spectra, in addition to the controllable light sources with visible and near‐infrared wavelengths. As a reference heart rate, a photoplethysmogram signal from an heart rate sensor is employed. Upon image acquisition, heart rate is estimated based on the facial images with varying intensities of visible and near‐infrared light sources under dim light conditions (10–50 lx). Compared to the values obtained using the visible light source alone, the signal‐to‐noise of the extracted signal increases and the root mean square error of the estimated heart rate decreases when the dual visible and near‐infrared light is applied. This study demonstrates that the use of dual visible and near‐infrared light sources can enhance the performance of non‐contact heart rate measurements, which could be applied to monitor the driver's status under dim light conditions.