Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis.

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Rapid Visual Detection of Spiroplasma eriocheiris by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye.

In recent years, a new type of Spiroplasma has been found that can cause "tremor disease" of the Chinese mitten crab Eriocheir sinensis. The outbreak of epidemic tremor disease has caused a serious setback in the Chinese mitten crab farming industry, with an incidence rate of more than 30% and mortality rates of 80-100%. Therefore, finding a sensitive method to detect tremor disease in E. sinensis has become a current research focus. In this research, a loop-mediated isothermal amplification detection method coupled with hydroxynaphthol blue dye (LAMP-HNB) was developed and used to rapidly detect Spiroplasma eriocheiris. First, we designed and synthesized specific outer primers, inner primers and loop primers based on the 16S ribosomal RNA gene of S. eriocheiris. Second, the LAMP-HNB detection method for S. eriocheiris was successfully established by screening the primers, adjusting the temperature and time of the reaction, and optimizing the concentrations of Mg2+ and dNTPs. In the specific tests, only samples infected with S. eriocheiris showed positive results, and other infections caused by bacteria and parasites tested negative, proving that the test has high specificity. Moreover, the detection limit was 2.5 × 10-6 ng/µL, indicating high sensitivity. This method for detecting S. eriocheiris provides rapid visual output based on LAMP-HNB detection and is a simple, fast, sensitive, and inexpensive method that can be applied to a wide range of field investigations.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1ß, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

Single-Shot Multi-Frame Imaging of Femtosecond Laser-Induced Plasma Propagation.

Single-shot ultrafast multi-frame imaging technology plays a crucial role in the observation of laser-induced plasma. However, there are many challenges in the application of laser processing, such as technology fusion and imaging stability. To provide a stable and reliable observation method, we propose an ultrafast single-shot multi-frame imaging technology based on wavelength polarization multiplexing. Through the frequency doubling and birefringence effects of the BBO and the quartz crystal, the 800 nm femtosecond laser pulse was frequency doubled to 400 nm, and a sequence of probe sub-pulses with dual-wavelength and different polarization was generated. The coaxial propagation and framing imaging of multi-frequency pulses provided stable imaging quality and clarity, as well as high temporal/spatial resolution (200 fs and 228 lp/mm). In the experiments involving femtosecond laser-induced plasma propagation, the probe sub-pulses measured their time intervals by capturing the same results. Specifically, the measured time intervals were 200 fs between the same color pulses and 1 ps between the adjacent different. Finally, based on the obtained system time resolution, we observed and revealed the evolution mechanism of femtosecond laser-induced air plasma filaments, the multifilament propagation of femtosecond laser in fused silica, and the influence mechanism of air ionization on laser-induced shock waves.

Development of Two Recombinase Polymerase Amplification EXO (RPA-EXO) and Lateral Flow Dipstick (RPA-LFD) Techniques for the Rapid Visual Detection of Aeromonas salmonicida.

Aeromonas salmonicida is the pathogen underlying furunculosis, causing a septicemic infection that influences both salmonid and non-salmonid fish. Early diagnosis of these contagions is essential for disease surveillance and prevention, so a rapid and sensitive approach is needed. Herein, a recombinase polymerase amplification EXO (RPA-EXO) assay and RPA with a lateral flow dipstick (RPA-LFD) were produced for testing A. salmonicida. The RPA-EXO and RPA-LFD primer sets were devised based on the conserved fragment sequence of the vapA gene. Then, RPA-EXO and RPA-LFD reaction systems were established, and the reaction temperature and time were optimized. After optimization, the RPA-EXO method was capable of testing A. salmonicida within 10 min, and the RPA-LFD method could detect A. salmonicida in only 5 min. The RPA-EXO and RPA-LFD methods exhibited high specificity with no cross-reaction with other strains. To assess sensitivity, a partial vapA gene was cloned, and serial plasmid dilutions were created ranging from 1 × 106 to 1 × 10-1 copies/µL. The detection limit of RPA-EXO was 1 × 102 copies/µL, and the detection limit of RPA-LFD was 1 copy/µL. For spiked turbot tissue samples, the sensitivity detection of A. salmonicida was 1.2 × 101 CFU/mL and 1.2 CFU/mL by RPA-EXO and RPA-LFD, respectively. In comparative analyses of clinical samples, the diagnostic results of RPA-EXO and RPA-LFD were compared with those of the standard conventional PCR test and showed nearly 100% consistency. Therefore, our RPA-EXO and RPA-LFD assays exhibited excellent specificity and sensitivity, which provided two simple, fast and dependable methods to conduct large-scale field investigations of A. salmonicida in resource-limited settings.

Immune responses to Vibrio vulnificus formalin-killed vaccine and ghost vaccine in Scophthalmus maximus.

In this research, Vibrio vulnificus formalin-killed (FKCs) vaccine and ghost (VVGs) vaccine were successfully developed, and shown to prevent vibriosis of Scophthalmus maximus resulting from V. vulnificus. The antibody titre of FKCs and VVGs vaccine was 1: 28 and 1: 211 . The RPS of FKCs and VVGs vaccine was 60% and 80%. In order to improve the understanding of vaccine protection mechanism, transcriptome data was used to analyse the immune response of S. maximus infected with V. vulnificus after vaccination with FKCs and VVGs vaccine. In the SmCon and SmIV groups, a series of innate immune-related genes were upregulated (such as, TLR5, Tp12, AP-1 and IL-1ß) or downregulated (such as, CASP6 and CASP8), which suggested that the immune protection mechanism induced by inactivated vaccine was similar to that of autoimmune response. In the SmIV and SmGho group, a number of innate and adaptive immune-related genes (such as, STAT1, IFN-γ and MHC Ia) were activated, in which the expression of these genes was higher in SmGho, and VVGs vaccine induced stronger innate and acquired immune responses. In conclusion, the results lay a foundation for further study on the molecular mechanisms of immune protection induced by VVGs vaccine and FKCs vaccine.

Throughput Improvement in Femtosecond Laser Ablation of Nickel by Double Pulses.

In this study, femtosecond laser double pulses were tested to improve their nickel ablation efficiency. The experimental results indicated that compared with single pulses, double pulses with different delay times generated craters with larger diameters and depths. The results obtained for three sets of double pulses with different energy ratios indicated that double pulses with an energy ratio of 1:9 had the highest ablation efficiency, followed by those with energy ratios of 2:8 and 5:5. The double pulses with the aforementioned three energy ratios achieved the maximum ablation efficiency when the delay time was 3-4 ps. Compared with single pulses, double pulses with an energy ratio of 1:9 generated craters with an up to 34% greater depth and up to 14% larger diameter. In addition, an interference effect was observed with a double pulse delay time of 0 ps, which has seldom been reported in the literature. The double pulses were simulated using the two-temperature model. The simulation results indicated that double pulses with an energy ratio of 1:9 with a delay time of 4 ps can perform the strongest ablation. These simulation results are in line with the experimental results.

Surface wave manipulation by plasmonic metasurface based on mode resonance.

We proposed a method to manipulate the surface waves with a deep subwavelength metasurface by applying resonators with interfering mode resonance. The simulation results demonstrate that a single deep subwavelength obstructed groove can effectively control the propagation of surface terahertz (THz) waves by a small step increase (< 1/20 λ) of the depth or a slight change of refractive index (Δn = 0.1). The surface waves transmitted and reflected by the single groove can be controlled periodically by increasing the groove depth or refractive index with a high efficiency owing to the mode resonance between surface spoof plasmonics modes and groove cavity modes. The generated circle resonance mode provides a new idea for the development of THz devices. Importantly, the transmitted or reflected intensity of the surface wave is also enhanced by the Mode resonance. It is a simple and effective method to operate surface THz waves and manufacture more compact integrated optical devices in deep subwavelength scale.

Effects of Flow-Induced Microfluidic Chip Wall Deformation on Imaging Flow Cytometry.

Imaging flow cytometry is a powerful tool by virtue of its capability for high-throughput cell analysis. The advent of high-speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow-induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 µm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X-Y plane and a significantly narrower stream in the Z-axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time-stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry.

Beam Manipulation Mechanisms of Dielectric Metasurfaces.

Dielectric metasurfaces can achieve flexible beam manipulations. Herein, we study dielectric metasurfaces with different refractive indices, periods, incident angles, and cross-sectional shapes to determine the metasurface working mechanisms. Perfect transmission mainly depends on multipolar interference that can be used to control the transmission modes through the hybrid periods, hybrid cross sections, and multilayers. Perfect reflection is strongly influenced by the period of the metasurface and occurs only when the period is shorter than incident wavelength, which can be attributed to the lattice coupling. Furthermore, lattice coupling can be classified into two types with distinct properties: vertical mode and horizontal mode coupling. The vertical mode appears when the effective wavelength matches the feature size, whereas the horizontal mode only appears when the incident wavelength is close to the period. The horizontal mode is sensitive to the incident angle. The revealed functioning mechanisms enable further practical applications of metasurfaces.

Intelligent whole-blood imaging flow cytometry for simple, rapid, and cost-effective drug-susceptibility testing of leukemia.

Drug susceptibility (also called chemosensitivity) is an important criterion for developing a therapeutic strategy for various cancer types such as breast cancer and leukemia. Recently, functional assays such as high-content screening together with genomic analysis have been shown to be effective for predicting drug susceptibility, but their clinical applicability is poor since they are time-consuming (several days long), labor-intensive, and costly. Here we present a highly simple, rapid, and cost-effective liquid biopsy for ex vivo drug-susceptibility testing of leukemia. The method is based on an extreme-throughput (>1 million cells per second), label-free, whole-blood imaging flow cytometer with a deep convolutional autoencoder, enabling image-based identification of the drug susceptibility of every single white blood cell in whole blood within 24 hours by simply flowing a drug-treated whole blood sample as little as 500 µL into the imaging flow cytometer without labeling. Our results show that the method accurately evaluates the drug susceptibility of white blood cells from untreated patients with acute lymphoblastic leukemia. Our method holds promise for affordable precision medicine.

Enhancing charge transfer with foreign molecules through femtosecond laser induced MoS2 defect sites for photoluminescence control and SERS enhancement.

Defect/active site control is crucial for tuning the chemical, optical, and electronic properties of MoS2, which can adjust the performance of MoS2 in application areas such as electronics, optics, catalysis, and molecular sensing. This study presents an effective method of inducing defect/active sites, including micro/nanofractured structures and S atomic vacancies, on monolayer MoS2 flakes by using femtosecond laser pulses, through which physical-chemical adsorption and charge transfer between foreign molecules (O2 or R6G molecules) and MoS2 are enhanced. The enhanced charge transfer between foreign molecules (O2 or R6G) and femtosecond laser-treated MoS2 can enhance the electronic doping effect between them, hence resulting in a photoluminescence photon energy shift (reaching 0.05 eV) of MoS2 and Raman enhancement (reaching 6.4 times) on MoS2 flakes for R6G molecule detection. Finally, photoluminescence control and micropatterns on MoS2 and surface-enhanced-Raman-scattering (SERS) enhancement of MoS2 for organic molecule detection are achieved. The proposed method, which can control the photoluminescence properties and arbitrary micropatterns on MoS2 and enhance its chemicobiological sensing performance for organic/biological molecules, has advantages of simplicity, maskless processing, strong controllability, high precision, and high flexibility, highlighting the superior ability of femtosecond laser pulses to achieve the property control and functionalization of two-dimensional materials.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

Label-free detection of cellular drug responses by high-throughput bright-field imaging and machine learning.

In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological change from different experiments can be inferred from the classification accuracy of a single classification model. Our work lays the groundwork for label-free drug screening in pharmaceutical science and industry.

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.