Multi-modal imaging using a cascaded microscope design.

We present a multi-modal fiber array snapshot technique (M-FAST) based on an array of 96 compact cameras placed behind a primary objective lens and a fiber bundle array. Our technique is capable of large-area, high-resolution, multi-channel video acquisition. The proposed design provides two key improvements to prior cascaded imaging system approaches: a novel optical arrangement that accommodates the use of planar camera arrays, and a new ability to acquire multi-modal image data acquisition. M-FAST is a multi-modal, scalable imaging system that can acquire snapshot dual-channel fluorescence images as well as differential phase contrast measurements over a large 6.59 mm × 9.74 mm field-of-view at 2.2-µm center full-pitch resolution.

Extreme UV Resist Exhibiting Synergism between Chemical and Physical Crosslinking Mechanisms.

Carbon-fluorine bonds in fluorinated molecules can undergo homolytic cleavage reactions when electrons are injected, and the resulting radicals combine to form network structures characterized by reduced solubility. This crosslinking chemistry suggests a new category of patterning materials that function under electron beam (e-beam) and extreme ultraviolet (EUV) lithographic conditions. Although this chemistry enables the production of 50 nm or smaller-sized features of simple fluoroalkylated polymers, it is limited by the need for relatively large amounts of irradiation energy to achieve required solubility changes. Therefore, this study was undertaken to devise a sensitivity-enhancing strategy based on a synergistic combination of radical crosslinking and hydrogen-bonding interactions between highly fluoroalkylated copolymers. An alternating copolymer was synthesized using tert-butoxystyrene and a fluoroalkylated maleimide, the former of which produces active hydrogens through catalytic acidolysis reactions. When the polymer was blended with a catalytic amount of a photoacid generator and subjected to lithographic patterning tests under e-beam and EUV irradiation, the deprotection reactions of tert-butoxy moieties proceeded at room temperature and led to a solubility decrease. We presume the small number of hydroxyl moieties produced formed an intermolecular hydrogen-bonding network, which acted synergistically with the covalent crosslinks generated by C-F bonds. When 30 nm features of copolymer thin films were fabricated by EUV lithography, sensitivity was improved by 25-34% without significant deterioration of pattern quality, especially line-edge roughness. These results demonstrate that EUV resists with improved patterning capabilities can be achieved by combining catalytic acidolysis reactions and noncatalytic crosslinking chemistry.

Intranasal trigeminal sensitivity to mechanical stimuli is associated with the perception of nasal patency.

PURPOSE: The aim of this prospective study was to examine the characteristics of a clinical test for the assessment of nasal trigeminal sensitivity to mechanical stimuli and its association with the perception of nasal patency. METHODS: Thirty-two normosmic healthy subjects participated (17 women and 15 men; age = 26 ± 3 years). Precisely defined air puffs were used with a flow rate of 2L/min for mechanical stimulation. They were presented to the nasal vestibule, nasal septum, and inferior turbinate with various stimulus durations. Thresholds were measured by single-staircase stimuli with changes in stimulus duration in steps of 10 ms. Trigeminal suprathreshold intensity was rated by subjects for stimulus durations of 200, 300, 400, and 500 ms. Test-retest reliability was examined by intraclass correlations (ICCs) and Bland-Altman plot with limits of agreement. Pearson's correlations were calculated between self-rated nasal patency and nasal trigeminal sensitivity. RESULTS: As indicated by trigeminal threshold and suprathreshold intensities, the nasal vestibule is the most sensitive area among the three locations, followed by the nasal septum and the inferior turbinate (p < 0.001). Coefficients of correlations between test and retest were 0.76 for thresholds, and 0.56 suprathreshold intensities (p < 0.001). The Bland-Altman analysis showed a good agreement between test-retest values. In addition, significant positive associations between trigeminal suprathreshold intensities and self-rated nasal obstruction were found at the inferior turbinate (r = 0.4, p < 0.05). CONCLUSION: Reliable assessment of nasal trigeminal sensitivity for air puffs appears to be possible. Nasal trigeminal suprathreshold sensitivity to mechanical stimuli is associated with the perception of nasal patency at the inferior turbinate. This opens a window into the assessment of the perception of nasal airflow in various clinical purposes, especially for patients with sinonasal diseases.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C-reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation-induced CRP. NOX inhibition using lentiviral sh-p22phox prevented apoptosis upon radiation-induced CRP. CRP overexpression reduced the amount of STAT3/Ref-1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref-1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co-overexpressed with Ref-1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP-Ref-1 complex. Immunofluorescence showed co-localization of CRP, Ref-1 and p53 with α-actin-positive VSMC in human atherosclerotic plaques. In conclusion, radiation-induced CRP increased the VSMCs apoptosis through Ref-1, which dissociated the STAT3/Ref-1 complex, interfered with JAK/STAT3 activity, and interacted with CRP-Ref-1, thus resulting in transcription-independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Increasing a microscope's effective field of view via overlapped imaging and machine learning.

This work demonstrates a multi-lens microscopic imaging system that overlaps multiple independent fields of view on a single sensor for high-efficiency automated specimen analysis. Automatic detection, classification and counting of various morphological features of interest is now a crucial component of both biomedical research and disease diagnosis. While convolutional neural networks (CNNs) have dramatically improved the accuracy of counting cells and sub-cellular features from acquired digital image data, the overall throughput is still typically hindered by the limited space-bandwidth product (SBP) of conventional microscopes. Here, we show both in simulation and experiment that overlapped imaging and co-designed analysis software can achieve accurate detection of diagnostically-relevant features for several applications, including counting of white blood cells and the malaria parasite, leading to multi-fold increase in detection and processing throughput with minimal reduction in accuracy.

3D printed transwell-integrated nose-on-chip model to evaluate effects of air flow-induced mechanical stresses on mucous secretion.

While there are many chip models that simulate the air-tissue interface of the respiratory system, only a few represent the upper respiratory system. These chips are restricted to unidirectional flow patterns that are not comparable to the highly dynamic and variable flow patterns found in the native nasal cavity. Here we describe the development of a tunable nose-on-chip device that mimics the air-mucosa interface and is coupled to an air delivery system that simulates natural breathing patterns through the generation of bi-directional air flow. Additionally, we employ computational modeling to demonstrate how the device design can be tuned to replicate desired mechanical characteristics within specific regions of the human nasal cavity. We also demonstrate how to culture human nasal epithelial cell line RPMI 2650 within the lab-on-chip (LOC) device. Lastly, Alcian Blue histological staining was performed to label mucin proteins, which play important roles in mucous secretion. Our results revealed that dynamic flow conditions can increase mucous secretion for RPMI 2650 cells, when compared to no flow, or stationary, conditions.

Maternal disease factors associated with neonatal jaundice: a case-control study.

BACKGROUND: Neonatal jaundice is common, and despite the considerable medical costs associated with it, there are still few studies on the maternal factors associated with it. Identification of maternal factors associated with neonatal jaundice is very important in terms of prevention, screening and management of neonatal jaundice. The current study aimed to identify maternal disease factors associated with neonatal jaundice. METHODS: We compared the maternal disease diagnostic codes during pregnancy (study A) and 1 year before conception (study B) in mothers whose insurance claims data included newborns treated for neonatal jaundice before birth registration via the National Health Insurance Service-National Sample Cohort (control group). To decrease the effect of confounding variables, the neonatal jaundice and control groups were matched at a ratio of 1:10 via propensity score matching using covariates including age and income. RESULTS: The matched samples for studies A and B included 4,026 and 3,278 (jaundice group: 366 and 298) delivery cases, respectively. In both studies, the jaundice group had a higher proportion of patients who underwent cesarean section than the control group. In study A, other diseases of the digestive system had the highest odds ratio (OR) (K92; adjusted OR: 14.12, 95% confidence interval [CI]: 2.70-82.26). Meanwhile, gastritis and duodenitis had the lowest OR (K29; adjusted OR: 0.39, 95% CI: 0.22-0.69). In study B, salpingitis and oophoritis had the highest OR (N70; adjusted OR: 3.33, 95% CI: 1.59-6.94). Heartburn had the lowest OR (R12; adjusted OR: 0.29, 95% CI:0.12-0.71). CONCLUSIONS: This study identified maternal disease factors correlated with neonatal jaundice during pregnancy and 1 year before conception. Maternal risk factors for neonatal jaundice included syphilis and leiomyoma during pregnancy, and salpingo-oophoritis before pregnancy. The protective factors included infection, inflammatory diseases, and dyspepsia.

Au Hierarchical Nanostructure-Based Surface Modification of Microelectrodes for Improved Neural Signal Recording.

Microelectrodes are widely used for neural signal analysis because they can record high-resolution signals. In general, the smaller the size of the microelectrode for obtaining a high-resolution signal, the higher the impedance and noise value of the electrodes. Therefore, to improve the signal-to-noise ratio (SNR) of neural signals, it is important to develop microelectrodes with low impedance and noise. In this research, an Au hierarchical nanostructure (AHN) was deposited to improve the electrochemical surface area (ECSA) of a microelectrode. Au nanostructures on different scales were deposited on the electrode surface in a hierarchical structure using an electrochemical deposition method. The AHN-modified microelectrode exhibited an average of 80% improvement in impedance compared to a bare microelectrode. Through electrochemical impedance spectroscopy analysis and impedance equivalent circuit modeling, the increase in the ECSA due to the AHN was confirmed. After evaluating the cell cytotoxicity of the AHN-modified microelectrode through an in vitro test, neural signals from rats were obtained in in vivo experiments. The AHN-modified microelectrode exhibited an approximate 9.79 dB improvement in SNR compared to the bare microelectrode. This surface modification technology is a post-treatment strategy used for existing fabricated electrodes, so it can be applied to microelectrode arrays and nerve electrodes made from various structures and materials.

Influence of metallization process on solution-processed InGaZnO thin film transistors.

Low-temperature solution-processed InGaZnO (IGZO) thin film transistors (TFTs) have recently attracted significant attention as the next-generation flexible display TFTs, owing to their high transparency, high electrical performance, low-cost fabrication, and large-area scalability. However, solution-processed amorphous IGZO TFTs have several drawbacks, such as poor film quality or low stability, and have been studied with view to improving the device performance. One of the critical components determining device characteristics is the metallization process, which we systematically studied using aluminum (Al) source and drain electrodes. The electrical properties were measured for different channel lengths and evaluated using the threshold voltage (Vth) and subthreshold swing (SS). Al electrodes directly affect the channel region, enhancing the electron density because of the doping effect from Al and oxygen vacancy-related oxidation of Al and causing an abnormal negative shift ofVth, which is confirmed by the component analysis via various spectroscopies. To understand and improve the TFT characteristics, we conducted a low-temperature post-annealing process and polymer passivation and succeeded in movingVthfrom over 150 V to near 0 V and remarkably improved SS. This study discovered that the influence of source-drain metallization on the channel region determines the device characteristics through the close relation between metal oxidation and the number of oxygen vacancies.

Multi-element microscope optimization by a learned sensing network with composite physical layers.

Standard microscopes offer a variety of settings to help improve the visibility of different specimens to the end microscope user. Increasingly, however, digital microscopes are used to capture images for automated interpretation by computer algorithms (e.g., for feature classification, detection, or segmentation), often without any human involvement. In this work, we investigate an approach to jointly optimize multiple microscope settings, together with a classification network, for improved performance with such automated tasks. We explore the interplay between optimization of programmable illumination and pupil transmission, using experimentally imaged blood smears for automated malaria parasite detection, to show that multi-element "learned sensing" outperforms its single-element counterpart. While not necessarily ideal for human interpretation, the network's resulting low-resolution microscope images (20X-comparable) offer a machine learning network sufficient contrast to match the classification performance of corresponding high-resolution imagery (100X-comparable), pointing a path toward accurate automation over large fields-of-view.

Highly Sensitive and Stretchable Resistive Strain Sensors Based on Microstructured Metal Nanowire/Elastomer Composite Films.

High sensitivity and high stretchability are two conflicting characteristics that are difficult to achieve simultaneously in elastic strain sensors. A highly sensitive and stretchable strain sensor comprising a microstructured metal nanowire (mNW)/elastomer composite film is presented. The surface structure is easily prepared by combining an mNW coating and soft-lithographic replication processes in a simple and reproducible manner. The densely packed microprism-array architecture of the composite film leads to a large morphological change in the mNW percolation network by efficiently concentrating the strain in the valley regions upon stretching. Meanwhile, the percolation network comprising mNWs with a high aspect ratio is stable enough to prevent electrical failure, even under high strains. This enables the sensor to simultaneously satisfy high sensitivity (gauge factor ≈81 at >130% strain) and high stretchability (150%) while ensuring long-term reliability (10 000 cycles at 150% strain). The sensor can also detect strain induced by bending and pressure, thus demonstrating its potential as a versatile sensing tool. The sensor is successfully utilized to monitor a wide range of human motions in real time. Furthermore, the unique sensing mechanism is easily extended to detect more complex multiaxial strains by optimizing the surface morphology of the device.

Role of interleukin-32 in the pathogenesis of endometriosis: in vitro, human and transgenic mouse data.

STUDY QUESTION: Does interleukin-32 (IL-32) play a role in the pathogenesis of endometriosis? SUMMARY ANSWER: IL-32 might be involved in the pathogenesis of endometriosis through increased viability, proliferation and invasion of endometrial cells. WHAT IS KNOWN ALREADY: Endometriosis is characterized as a chronic inflammatory disease and several proinflammatory cytokines are suggested to be involved in its pathogenesis and pathophysiology. IL-32, recognized as a new proinflammatory cytokine and a strong inducer of other proinflammatory cytokines, has been shown to serve as a key modulator in several chronic inflammatory diseases. STUDY DESIGN, SIZE, DURATION: This study included comparison of IL-32 levels in the peritoneal fluids between women with and without endometriosis, in-vitro experiments using Ishikawa cells and endometrial stromal cells (ESCs), and experiments on IL-32 transgenic mice and wild-type mice with induced endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: IL-32 levels in the peritoneal fluids were measured using enzyme-linked immunosorbent assays. Cell viability, expression of proliferating cell nuclear antigen (PCNA), and cellular invasiveness were analyzed following in-vitro treatment of Ishikawa cells and ESCs with recombinant IL-32 alpha (α) and gamma (γ). Ectopic endometriotic lesions were compared between IL-32 transgenic mice and wild-type mice after autologous endometrial transplantation with immunohistochemistry for Ki-67 antigen and PCNA. MAIN RESULTS AND THE ROLE OF CHANCE: The peritoneal fluid concentration of IL-32 was significantly higher in patients with advanced stage endometriosis compared with the controls. In-vitro treatment with IL-32 α and γ caused significant increases in cellular viability, PCNA expression, and invasiveness in Ishikawa cells and ESCs. The IL-32 transgenic mice had a significantly larger size of the ectopic endometrial lesions with higher expression of Ki-67 antigen and PCNA compared with wild-type mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: It is still unclear whether IL-32 is a main regulator, or one of several downstream proinflammatory cytokines, causing establishment and/or progression of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Further investigation on IL-32 signaling pathways may contribute to development a more effective treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI16C1682). None of the authors has anything to disclose.

Nasal Structural and Aerodynamic Features That May Benefit Normal Olfactory Sensitivity.

Nasal airflow that effectively transports ambient odors to the olfactory receptors is important for human olfaction. Yet, the impact of nasal anatomical variations on airflow pattern and olfactory function is not fully understood. In this study, 22 healthy volunteers were recruited and underwent computed tomographic scans for computational simulations of nasal airflow patterns. Unilateral odor detection thresholds (ODT) to l-carvone, phenylethyl alcohol (PEA) and d-limonene were also obtained for all participants. Significant normative variations in both nasal anatomy and aerodynamics were found. The most prominent was the formation of an anterior dorsal airflow vortex in some but not all subjects, with the vortex size being significantly correlated with ODT of l-carvone (r = 0.31, P < 0.05). The formation of the vortex is likely the result of anterior nasal morphology, with the vortex size varying significantly with the nasal index (ratio of the width and height of external nose, r = -0.59, P < 0.001) and nasal vestibule "notch" index (r = 0.76, P < 0.001). The "notch" is a narrowing of the upper nasal vestibule cartilage region. The degree of the notch also significantly correlates with ODT for PEA (r = 0.32, P < 0.05) and l-carvone (r = 0.33, P < 0.05). ODT of d-limonene, a low mucosal soluble odor, does not correlate with any of the anatomical or aerodynamic variables. The current study revealed that nasal anatomy and aerodynamics might have a significant impact on normal olfactory sensitivity, with greater airflow vortex and a narrower vestibule region likely intensifying the airflow vortex toward the olfactory region and resulting in greater olfactory sensitivity to high mucosal soluble odors.

Priming with ceramide-1 phosphate promotes the therapeutic effect of mesenchymal stem/stromal cells on pulmonary artery hypertension.

Some molecules enriched in damaged organs can contribute to tissue repair by stimulating the mobilization of stem cells. These so-called "priming" factors include bioactive lipids, complement components, and cationic peptides. However, their therapeutic significance remains to be determined. Here, we show that priming of mesenchymal stromal/stem cells (MSCs) with ceramide-1 phosphate (C1P), a bioactive lipid, enhances their therapeutic efficacy in pulmonary artery hypertension (PAH). Human bone marrow (BM)-derived MSCs treated with 100 or 200 µM C1P showed improved migration activity in Transwell assays compared with non-primed MSCs and concomitantly activated MAPK(p42/44) and AKT signaling cascades. Although C1P priming had little effect on cell surface marker phenotypes and the multipotency of MSCs, it potentiated their proliferative, colony-forming unit-fibroblast, and anti-inflammatory activities. In a monocrotaline-induced PAH animal model, a single administration of human MSCs primed with C1P significantly attenuated the PAH-related increase in right ventricular systolic pressure, right ventricular hypertrophy, and thickness of α-smooth muscle actin-positive cells around the vessel wall. Thus, this study shows that C1P priming increases the effects of MSC therapy by enhancing the migratory, self-renewal, and anti-inflammatory activity of MSCs and that MSC therapy optimized with priming protocols might be a promising option for the treatment of PAH patients.

A blockchain-based healthcare data marketplace: prototype and demonstration.

Objectives: This study aimed to develop healthcare data marketplace using blockchain-based B2C model that ensures the transaction of healthcare data among individuals, companies, and marketplaces. Materials and methods: We designed an architecture for the healthcare data marketplace using blockchain. A healthcare data marketplace was developed using Panacea, MySQL 8.0, JavaScript library, and Node.js. We evaluated the performance of the data marketplace system in 3 scenarios. Results: We developed mobile and web applications for healthcare data marketplace. The transaction data queries were executed fully within about 1-2 s, and approximately 9.5 healthcare data queries were processed per minute in each demonstration scenario. Discussion: Blockchain-based healthcare data marketplaces have shown compliance performance in the process of data collection and will provide a meaningful role in analyzing healthcare data. Conclusion: The healthcare data marketplace developed in this project can iron out time and place limitations and create a framework for gathering and analyzing fragmented healthcare data.

Recyclable Superhydrophobic Surface Prepared via Electrospinning and Electrospraying Using Waste Polyethylene Terephthalate for Self-Cleaning Applications.

Superhydrophobic surfaces, i.e., surfaces with a water contact angle (WCA) ≥ 150°, have gained much attention as they are multifunctional surfaces with features such as self-cleaning, which can be useful in various applications such as those requiring waterproof and/or protective films. In this study, we prepared a solution from recycled polyethylene terephthalate (PET) and fabricated a superhydrophobic surface using electrospinning and electrospraying processes. We observed that the fabricated geometry varies depending on the solution conditions, and based on this, we fabricated a hierarchical structure. From the results, the optimized structure exhibited a very high WCA (>156.6°). Additionally, our investigation into the self-cleaning functionality and solar panel efficiency of the fabricated surface revealed promising prospects for the production of superhydrophobic surfaces utilizing recycled PET, with potential applications as protective films for solar panels. Consequently, this research contributes significantly to the advancement of environmentally friendly processes and the progress of recycling technology.

3D Printing as a Planning Tool to Optimize Sinonasal Irrigation.

BACKGROUND: Topical sinus irrigation plays a critical role in the management of sinonasal diseases. Yet, the penetration of irrigant to targeted sinuses may be highly variable and difficult to predict. Here, we investigate the use of 3D printing as a planning tool to optimize outcomes. METHODS: Eight post-operative models were 3D printed with a FormLabs Form3 printer based on individual CT scans. Irrigations were performed and video recorded with a squeeze bottle attached via silicon water-tight seal, in 4 head positions: 45° to-the-side, 90° to-the-side, 45° forward and 45° to-the-side, and 90° forward, with irrigation fluid entering the upper (conventional) or lower (backfill) nostrils. RESULTS: Significant individual variations were observed in sinus penetration as a function of head position. In general, the maxillary sinus was the easiest to irrigate in most head positions (P < .05), followed by frontal and ethmoid, with sphenoid being the most difficult. Both the 90°-to-the-side and the 90°-forward positions were significantly more effective than the others (P < .05), with 90°-forward better for frontal sinuses and 90°-to-the-side superior for all other sinuses. The backfill was significantly superior to conventional technique in head positions involving a side tilt (P < .05). CONCLUSION: Variations in technique and position significantly impacted irrigation outcome. Backfill irrigation that pushes fluid against gravity to pool around the ostium, seems to provide overall better outcomes. This study demonstrates the advantage of 3D printing as a rapid planning tool to guide irrigation strategies.

Microheater with copper nanofiber network via electrospinning and electroless deposition.

In this report, we present the development of a copper nanofiber network-based microheater, designed for applications in electron microscopes, gas sensing, and cell culture platforms. The seed layer, essential for electroless deposition, was fabricated through the electrospinning of a palladium-contained polyvinylpyrrolidone solution followed by a heat treatment. This process minimized the contact resistance between nanofibers. We successfully fabricated a microheater with evenly distributed temperature by controlling the electrospinning time, heat treatment conditions, and electroless deposition time. We assessed the electrical and thermal characteristics of the microheater by examining the nanofiber density, sheet resistance, and transmittance. The microheater's performance was evaluated by applying current, and we verified its capacity to heat up to a maximum of 350 °C. We further observed the microheater's temperature distribution at varying current levels through an infrared camera. The entire manufacturing procedure takes place under normal pressure, eliminating the need for masking or etching processes. This renders the method easily adaptable to the mass production of microdevices. The method is expected to be applicable to various materials and sizes and is cost-effective compared to commercially produced microheaters developed through microelectromechanical system processes, which demand complex facilities and high cost.

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.

Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.

Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.