Microfluidic Impedance Cytometry Enabled One-Step Sample Preparation for Efficient Single-Cell Mass Spectrometry.

Single-cell mass spectrometry (MS) is significant in biochemical analysis and holds great potential in biomedical applications. Efficient sample preparation like sorting (i.e., separating target cells from the mixed population) and desalting (i.e., moving the cells off non-volatile salt solution) is urgently required in single-cell MS. However, traditional sample preparation methods suffer from complicated operation with various apparatus, or insufficient performance. Herein, a one-step sample preparation strategy by leveraging label-free impedance flow cytometry (IFC) based microfluidics is proposed. Specifically, the IFC framework to characterize and sort single-cells is adopted. Simultaneously with sorting, the target cell is transferred from the local high-salinity buffer to the MS-compatible solution. In this way, one-step sorting and desalting are achieved and the collected cells can be directly fed for MS analysis. A high sorting efficiency (>99%), cancer cell purity (≈87%), and desalting efficiency (>99%), and the whole workflow of impedance-based separation and MS analysis of normal cells (MCF-10A) and cancer cells (MDA-MB-468) are verified. As a standalone sample preparation module, the microfluidic chip is compatible with a variety of MS analysis methods, and envisioned to provide a new paradigm in efficient MS sample preparation, and further in multi-modal (i.e., electrical and metabolic) characterization of single-cells.

Capillarity Enabled Large-Array Liquid Metal Electrodes for Compact and High-Throughput Dielectrophoretic Microfluidics.

Dielectrophoresis (DEP) particle separation has label-free, well-controllable, and low-damage merits. Sidewall microelectrodes made of liquid metal alloy (LMA) inherits the additional advantage of thick electrodes to generate impactful DEP force. However, existing LMA electrode-based devices lack the ability to integrate large-array electrodes in a compact footprint, severely limiting flow rate and thus throughput. Herein, a facile and versatile method is proposed to integrate high-density thick LMA electrodes in microfluidic devices, taking advantage of the passive control ability of capillary burst valves (CBVs). CBVs with carefully designed burst pressures are co-designed in microfluidic channels, allowing self-assembly of LMA electrode array through simple hand-push injection. The arrayed electrode configuration brings the accumulative DEP deflection effect. Specifically, The fabricated 5000 pairs of sidewall electrodes in a compact chip are demonstrted to achieve ten times higher throughput in DEP deflection. The 5000-electrode-pair device is applied to successfully separate four mixed samples, including human peripheral blood mononuclear cells and A549 cells with the flow rate of 70 µL min-1 . It is envisioned that this work can greatly facilitate LMA electrode array fabrication and offer a robust and versatile platform for DEP separation applications.

Impedance-Based Multimodal Electrical-Mechanical Intrinsic Flow Cytometry.

Reflecting various physiological states and phenotypes of single cells, intrinsic biophysical characteristics (e.g., mechanical and electrical properties) are reliable and important, label-free biomarkers for characterizing single cells. However, single-modal mechanical or electrical properties alone are not specific enough to characterize single cells accurately, and it has been long and challenging to couple the conventionally image-based mechanical characterization and impedance-based electrical characterization. In this work, the spatial-temporal characteristics of impedance sensing signal are leveraged, and an impedance-based multimodal electrical-mechanical flow cytometry framework for on-the-fly high-dimensional intrinsic measurement is proposed, that is, Young's modulus E, fluidity ß, radius r, cytoplasm conductivity σi , and specific membrane capacitance Csm , of single cells. With multimodal high-dimensional characterization, the electrical-mechanical flow cytometry can better reveal the difference in cell types, demonstrated by the experimental results with three types of cancer cells (HepG2, MCF-7, and MDA-MB-468) with 93.4% classification accuracy and pharmacological perturbations of the cytoskeleton (fixed and Cytochalasin B treated cells) with 95.1% classification accuracy. It is envisioned that multimodal electrical-mechanical flow cytometry provides a new perspective for accurate label-free single-cell intrinsic characterization.

Evaluating the Accuracy of Impedance Flow Cytometry with Cell-Sized Liposomes.

Electrical properties of single cells are important label-free biomarkers of disease and immunity. At present, impedance flow cytometry (IFC) provides important means for high throughput characterization of single-cell electrical properties. However, the accuracy of the spherical single-shell electrical model widely used in IFC has not been well evaluated due to the lack of reliable and reproducible single-shell model particles with true-value electrical parameters as benchmarks. Herein, a method is proposed to evaluate the accuracy of the single-cell electrical model with cell-sized unilamellar liposomes synthesized through double emulsion droplet microfluidics. The influence of three key dimension parameters (i.e., the measurement channel width w, height h, and electrode gap g) in the single-cell electrical model were evaluated through experiment. It was found that the relative error of the electrical intrinsic parameters measured by IFC is less than 10% when the size of the sensing zone is close to the measured particles. It further reveals that h has the greatest influence on the measurement accuracy, and the maximum relative error can reach ∼30%. Error caused by g is slightly larger than w. This provides a solid guideline for the design of IFC measurement system. It is envisioned that this method can advance further improvement of IFC and accurate electrical characterization of single cells.

High-Efficiency Single-Cell Electrical Impedance Spectroscopy.

Single-cell impedance measurement is label free and noninvasive in characterizing the electrical properties of single cells. At present, though widely used for impedance measurement, electrical impedance flow cytometry (IFC) and electrical impedance spectroscopy (EIS) are used alone for most microfluidic chips. Here, we describe high-efficiency single-cell electrical impedance spectroscopy, which combines in one chip the IFC and EIS techniques for high-efficiency single-cell electrical property measurement. We envision that the strategy of combining IFC and EIS provides a new thought in the efforts to enhance the efficiency of electrical property measurement for single cells.

Performance-enhanced clogging-free viscous sheath constriction impedance flow cytometry.

As a label-free and high-throughput single cell analysis platform, impedance flow cytometry (IFC) suffers from clogging caused by a narrow microchannel as mechanical constriction (MC). Current sheath constriction (SC) solutions lack systematic evaluation of the performance and proper guidelines for the sheath fluid. Herein, we hypothesize that the viscosity of the non-conductive liquid is the key to the performance of SC, and propose to employ non-conductive viscous sheath flow in SC to unlock the tradeoff between sensitivity and throughput, while ensuring measurement accuracy. By placing MC and SC in series in the same microfluidic chip, we established an evaluation platform to prove the hypothesis. Through modeling analysis and experiments, we confirmed the accuracy (error < 1.60% ± 4.71%) of SC w.r.t. MC, and demonstrated that viscous non-conductive PEG solution achieved an improved sensitivity (7.92×) and signal-to-noise ratio (1.42×) in impedance measurement, with the accuracy maintained and free of clogging. Viscous SC IFC also shows satisfactory ability to distinguish different types of cancer cells and different subtypes of human breast cancer cells. It is envisioned that viscous SC IFC paves the way for IFC to be really usable in practice with clogging-free, accurate, and sensitive performance.

Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry.

Mass spectrometry (MS) has become a powerful tool for metabolome, lipidome, and proteome analyses. The efficient analysis of multi-omics in single cells, however, is still challenging in the manipulation of single cells and lack of in-fly cellular digestion and extraction approaches. Here, we present a streamlined strategy for highly efficient and automatic single-cell multi-omics analysis by MS. We developed a 10-pL-level microwell chip for housing individual single cells, whose proteins were found to be digested in 5 min, which is 144 times shorter than traditional bulk digestion. Besides, an automated picoliter extraction system was developed for sampling of metabolites, phospholipids, and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell sample. In addition, 1391 proteins, phospholipids, and metabolites were detected from one single cell within 10 min. We further analyzed cells digested from cancer tissue samples, achieving up to 40% increase in cell classification accuracy using multi-omics analysis in comparison with single-omics analysis. This automated single-cell MS strategy is highly efficient in analyzing multi-omics information for investigation of cell heterogeneity and phenotyping for biomedical applications.

Event-based Semantic Segmentation with Posterior Attention.

In the past years, attention-based Transformers have swept across the field of computer vision, starting a new stage of backbones in semantic segmentation. Nevertheless, semantic segmentation under poor light conditions remains an open problem. Moreover, most papers about semantic segmentation work on images produced by commodity frame-based cameras with a limited framerate, hindering their deployment to auto-driving systems that require instant perception and response at milliseconds. An event camera is a new sensor that generates event data at microseconds and can work in poor light conditions with a high dynamic range. It looks promising to leverage event cameras to enable perception where commodity cameras are incompetent, but algorithms for event data are far from mature. Pioneering researchers stack event data as frames so that event-based segmentation is converted to framebased segmentation, but characteristics of event data are not explored. Noticing that event data naturally highlight moving objects, we propose a posterior attention module that adjusts the standard attention by the prior knowledge provided by event data. The posterior attention module can be readily plugged into many segmentation backbones. Plugging the posterior attention module into a recently proposed SegFormer network, we get EvSegFormer (the event-based version of SegFormer) with state-of-the-art performance in two datasets (MVSEC and DDD-17) collected for event-based segmentation. Code is available at https://github.com/zexiJia/EvSegFormer to facilitate research on event-based vision.

Non-Invasive and Minute-Frequency 3D Tomographic Imaging Enabling Long-Term Spatiotemporal Observation of Single Cell Fate.

Non-invasive and rapid imaging technique at subcellular resolution is significantly important for multiple biological applications such as cell fate study. Label-free refractive-index (RI)-based 3D tomographic imaging constitutes an excellent candidate for 3D imaging of cellular structures, but its full potential in long-term spatiotemporal cell fate observation is locked due to the lack of an efficient integrated system. Here, a long-term 3D RI imaging system incorporating a cutting-edge white light diffraction phase microscopy module with spatiotemporal stability, and an acoustofluidic device to roll and culture single cells in a customized live cell culture chamber is reported. Using this system, 3D RI imaging experiments are conducted for 250 cells and demonstrate efficient cell identification with high accuracy. Importantly, long-term and frequency-on-demand 3D RI imaging of K562 and MCF-7 cancer cells reveal different characteristics during normal cell growth, drug-induced cell apoptosis, and necrosis of drug-treated cells. Overall, it is believed that the proposed 3D tomographic imaging technique opens up a new avenue for visualizing intracellular structures and will find many applications such as disease diagnosis and nanomedicine.

Floating-Electrode-Enabled Impedance Cytometry for Single-Cell 3D Localization.

As a label-free, low-cost, and noninvasive tool, impedance measurement has been widely used in single-cell characterization analysis. However, due to the tiny volume of cells, the uncertainty of the spatial position in the microchannel will bring measurement errors in single-cell electrical parameters. To overcome the issue, we designed a novel microdevice configured with a coplanar differential electrode structure to accurately resolve the spatial position of single cells without constraining techniques such as additional sheath fluids or narrow microchannels. The device precisely localizes single cells by measuring the induced current generated by the combined action of the floating electrode and the differential electrodes when single cells flow through the electrode-sensing area. The device was experimentally validated by measuring 6 µm yeast cells and 10 µm particles, achieving spatial localization with a resolution down to 2.1 µm (about 5.3% of the channel width) in lateral direction and 1.2 µm (about 5.9% of the channel height) in the vertical direction at a flow rate of 1.2 µL/min. In addition, by comparing measurement of yeast cells and particles, it was demonstrated that the device not only localizes the single cells or particles but also simultaneously characterizes their status properties such as velocity and size. The device offers a competitive electrode configuration in impedance cytometry with the advantages of simple structure, low cost, and high throughput, promising cell localization and thus electrical characterization.

Automated and Miniaturized Pico-Liter Metabolite Extraction System for Single-Cell Mass Spectrometry.

OBJECTIVE: Mass spectrometry has become the method of choice for single cell analysis due to its high sensitivity of detection and capability in analyzing a large number of metabolites simultaneously. For a long time, an automated and miniaturized system capable of extracting cellular contents from single cells at the pico-liter level for pico-ESI analysis has been lacking. METHODS: This paper presents a first-of-its-kind automated and miniaturized pico-liter extraction system for single-cell MS. The key modules, including imaging, bus controller, and fluidic driving are customized to achieve satisfactory performance at affordable costs, resulting in a miniaturized system movable on a trolley and connectable with the MS. To enable automation, a single cell trapping device, new image-based one-pixel accuracy positioning methods for cells and micropipette, and a surface-tension-based 1-pL accuracy volume control scheme are developed. RESULTS: The system is able to control the solvent loading at 1.97 ± 0.05 nL, solvent dispensing at 14-15 pL, and solvent evaporation at 689±48 pL. MS experiments demonstrate a throughput of 20 cells/h. CONCLUSION: The system has achieved better performance in consistency (∼21%), sensitivity (∼28%), and success rate (up to 40%) than manual operation. SIGNIFICANCE: This automated and miniaturized system lays a solid basis for applying pico-ESI MS analysis in the automated and high-throughput single cell MS analysis, such as single-cell metabolomics and lipidomics.

The effect of thermodynamic changes in the cooling of saline soils on the corrosion system of carbon steels.

In this experiment, Q235 and X80 carbon steels, which are widely used in oil and gas pipelines and ancillary facilities, were selected to study the changes in the corrosion behaviour and mechanism of carbon steels in the process of natural saline soil cooling to a freezing state through electrochemical testing. The equivalent circuit model of carbon steel before and after the freezing phase transformation in the soil was determined. Based on the corrosion kinetic parameters and soil thermodynamic changes, the influencing factors of steel corrosion during the cooling process were systematically analysed. It was found that temperature mainly affected carbon steel corrosion by changing the properties of the solution. The main factors affecting the corrosion behaviour of the carbon steel were the thermal motion of molecules, ions, and electrons in solution, oxygen dissolution and diffusion, ion adsorption, diffusion mass transfer, and unfrozen water content change during the cooling process.

Dual-Path Effect of Mortality Salience Induced by COVID-19 on Food Safety Behavior in China.

During the pandemic, the mortality salience of COVID-19 has affected everyone. The public is extremely sensitive to food safety, especially cold chain food and imported food. This research is based on the terror management theory, protective motivation theory, and self-construal theory. It proposes an integrated dual-path framework to explore the different mechanisms that mortality salience has on food safety behavior. The result of three experiments verified our conjectures. First, mortality salience positively affects individuals' food safety behavior. More importantly, we found the dual-path mechanism that underlies the effect, that is, the mediating of self-protective motivation and prosocial motivation. In addition, different self-construals make the confirmed effect clear. These findings provide implications for the government to protect public food safety and health.

Impedance-Enabled Camera-Free Intrinsic Mechanical Cytometry.

Mechanical properties of single cells are important label-free biomarkers normally measured by expensive and complex imaging systems. To unlock this limit and allow mechanical properties comparable across different measurement platforms, camera-free intrinsic mechanical cytometry (CFIMC) is proposed for on-the-fly measurement of two major intrinsic mechanical parameters, that is, Young's modulus E and fluidity ß, of single cells. CFIMC adopts a framework that couples the impedance electrodes with the constriction channel spatially, so that the impedance signals contain the dynamic deformability information of the cell squeezing through the constriction channel. Deformation of the cell is thus extracted from the impedance signals and used to derive the intrinsic mechanical parameters. With reasonably high throughput (>500 cells min-1 ), CFIMC can successfully reveal the mechanical difference in cancer and normal cells (i.e., human breast cell lines MCF-10A, MCF-7, and MDA-MB-231), living and fixed cells, and pharmacological perturbations of the cytoskeleton. It is further found that 1 µM level concentration of Cytochalasin B may be the threshold for the treated cells to induce a significant cytoskeleton effect reflected by the mechanical parameters. It is envisioned that CFIMC provides an alternative avenue for high-throughput and real-time single-cell intrinsic mechanical analysis.

Electrochemical Corrosion Behaviour of X70 Steel under the Action of Capillary Water in Saline Soils.

In this paper, the electrochemical corrosion behavior of X70 steel in saline soil under capillary water was simulated by a Geo-experts one-dimensional soil column instrument. A volumetric water content sensor and conductivity test were used to study the migration mechanism of water and salt (sodium chloride) under the capillary water. The electrochemical corrosion behavior of the X70 steel in the corrosion system was analyzed by electrochemical testing as well as the macroscopic and microscopic corrosion morphology of the steel. The test results showed that the corrosion behavior of X70 steel was significantly influenced by the rise of capillary water. In particular, the wetting front during the capillary water rise meant that the X70 steel was located at the three-phase solid/liquid/gas interface at a certain location, which worsened its corrosion behavior. In addition, after the capillary water was stabilized, the salts were transported with the capillary water to the top of the soil column. This resulted in the highest salt content in the soil environment and the most severe corrosion of the X70 steel at this location.

High content drug screening of primary cardiomyocytes based on microfluidics and real-time ultra-large-scale high-resolution imaging.

High content screening (HCS) plays an important role in biological applications and drug development. Existing techniques fail to simultaneously meet multiple needs of throughput, efficiency in sample and chemical consumption, and real-time imaging of a large view at high resolution. Leveraging advances in microfluidics and imaging technology, we setup a new paradigm of large-scale, high-content drug screening solutions for rapid biological processes, like cardiotoxicity. The designed microfluidic chips enable 10 types of drugs each with 5 concentrations to be assayed simultaneously. The imaging system has 30 Hz video rate for a centimeter filed-of-view at 0.8 µm resolution. Using the HCS system, we assayed 12 small molecules through their effects on the Ca2+ ion signal of cardiomyocytes. Experimental results demonstrated the unparalleled capability of the system in revealing the spatiotemporal patterns of Ca2+ imaging of cardiomyocytes, and validated the cardiotoxicity of certain molecules. We envision that this new HCS paradigm and cutting-edge platform offer the most advanced alternative to well-plate based methods.

Neural network-enhanced real-time impedance flow cytometry for single-cell intrinsic characterization.

Single-cell impedance flow cytometry (IFC) is emerging as a label-free and non-invasive method for characterizing the electrical properties and revealing sample heterogeneity. At present, most IFC studies utilize phenomenological parameters (e.g., impedance amplitude, phase and opacity) to characterize single cells instead of intrinsic biophysical metrics (e.g., radius r, cytoplasm conductivity σi and specific membrane capacitance Csm). Intrinsic parameters are normally calculated off-line by time-consuming model-fitting methods. Here, we propose to employ neural network (NN)-enhanced IFC to achieve both real-time single-cell intrinsic characterization and intrinsic parameter-based cell classification at high throughput. Three intrinsic parameters (r, σi and Csm) can be obtained online and in real-time via a trained NN at 0.3 ms per single-cell event, achieving significant improvement in calculation speed. Experiments involving four cancer cells and one lymphocyte cell demonstrated 91.5% classification accuracy in the cell type for a test group of 9751 cell samples. By performing a viability assay, we provide evidence that the IFC test per se would not substantially affect the cell property. We envision that the NN-enhanced real-time IFC will provide a new platform for high-throughput, real-time and online cell intrinsic electrical characterization.

Does a Large Ear Type Wheat Variety Benefit More From Elevated CO2 Than That From Small Multiple Ear-Type in the Quantum Efficiency of PSII Photochemistry?

Recently, several reports have suggested that the growth and grain yield of wheat are significantly influenced by high atmospheric carbon dioxide concentration (CO2) because of it photosynthesis enhancing effects. Moreover, it has been proposed that plants with large carbon sink size will benefit more from CO2 enrichment than those with small carbon sink size. However, this hypothesis is yet to be test in winter wheat plant. Therefore, the aim of this study was to examine the effect of elevated CO2 (eCO2) conditions on the quantum efficiency of photosystem II (PSII) photochemistry in large ear-type (cv. Shanhan 8675; greater ear C sink strength) and small multiple ear-type (cv. Early premium; greater vegetative C source strength) winter wheat varieties. The experiment was conducted in a free air CO2 enrichment (FACE) facility, and three de-excitation pathways of the primary reaction of PSII of flag leaf at the anthesis stage were evaluated under two CO2 concentrations (ambient [CO2], ∼415 µmol⋅mol-1, elevated [CO2], ∼550 µmol⋅mol-1) using a non-destructive technique of modulated chlorophyll fluorescence. Additionally, the grain yield of the two varieties was determined at maturity. Although elevated CO2 increased the quantum efficiency of PSII photochemistry (ΦPSII) of Shanhan 8675 (SH8675) flag leaves at the anthesis stage, the grain number per ear and 1,000-kernel weight were not significantly affected. In contrast, the ΦPSII of early premium (ZYM) flag leaves was significantly lower than that of SH8675 flag leaves at the anthesis stage, which was caused by an increase in the regulatory non-photochemical energy dissipation quantum (ΦNPQ) of PSII, suggesting that light energy absorbed by PSII in ZYM flag leaf was largely dissipated as thermal energy. The findings of our study showed that although SH8675 flag leaves exhibited higher C sink strength and quantum efficiency of PSII photochemistry at the anthesis stage, these factors alone do not ensure improved grain yield under eCO2 conditions.

A microfluidic device enabling deterministic single cell trapping and release.

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive microfluidic devices cannot deterministically release trapped cells for further analysis. In this paper, we present a novel microfluidic device that can achieve high-efficiency cell trapping, which can then be released in a deterministic order. The device contains an array of trapping sites, a main channel, a trigger channel, and an air channel. Two types of capillary valves are configured along the channels. As these capillary valves can be automatically opened in a predefined pattern, the incoming cells can be spontaneously and sequentially trapped into separate trapping sites. After trapping, the individual trapped cells can be released from their sites in a last-trapped-first-released manner by applying pressure from the trigger channel to counteract against the pressure from the main channel. The theoretical model of the trapping and release flow field is established respectively to describe the conditions required for trapping and release. Experiments using MCF-7 cells demonstrated the capability of our device for deterministic single cell trapping and release. We envision that our method constitutes a useful sample preparation platform for single cell analysis.

Dual-fiber microfluidic chip for multimodal manipulation of single cells.

On-chip single-cell manipulation is imperative in cell biology and it is desirable for a microfluidic chip to have multimodal manipulation capability. Here, we embedded two counter-propagating optical fibers into the microfluidic chip and configured their relative position in space to produce different misalignments. By doing so, we demonstrated multimodal manipulation of single cells, including capture, stretching, translation, orbital revolution, and spin rotation. The rotational manipulation can be in-plane or out-of-plane, providing flexibility and capability to observe the cells from different angles. Based on out-of-plane rotation, we performed a 3D reconstruction of cell morphology and extracted its five geometric parameters as biophysical features. We envision that this type of microfluidic chip configured with dual optical fibers can be helpful in manipulating cells as the upstream process of single-cell analysis.