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1.
Electrophoresis ; 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34599837

##### RESUMO

Droplet microfluidics has emerged as a powerful tool for a diverse range of biomedical and industrial applications such as single-cell analysis, directed evolution, and metabolic engineering. In these applications, droplet sorting has been effective for isolating small droplets encapsulating molecules, cells, or crystals of interest. Recently, there is an increased interest in extending the applicability of droplet sorting to larger droplets to utilize their size advantage. However, sorting throughputs of large droplets have been limited, hampering their wide adoption. Here, we report our demonstration of high-throughput fluorescence-activated droplet sorting of 1 nL droplets using an upgraded version of the sequentially addressable dielectrophoretic array (SADA), which we reported previously. The SADA is an array of electrodes that are individually and sequentially activated/deactivated according to the speed and position of a droplet passing nearby the array. We upgraded the SADA by increasing the number of driving electrodes constituting the SADA and incorporating a slanted microchannel. By using a ten-electrode SADA with the slanted microchannel, we achieved fluorescence-activated droplet sorting of 1 nL droplets at a record high throughput of 1752 droplets/s, twice as high as the previously reported maximum sorting throughput of 1 nL droplets.

2.
Opt Lett ; 46(17): 4320-4323, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470004

##### RESUMO

We report highly sensitive Fourier-transform coherent anti-Stokes Raman scattering spectroscopy enabled by genetic algorithm (GA) pulse shaping for adaptive dispersion compensation. We show that the non-resonant four-wave mixing signal from water can be used as a fitness indicator for successful GA training. This method allows GA adaptation to sample measurement conditions and offers significantly improved performance compared to training using second-harmonic generation from a nonlinear crystal in place of the sample. Results include a 3× improvement to peak signal-to-noise ratio for 2-propanol measurement, as well as a 10× improvement to peak intensities from the high-throughput measurement of polystyrene microbeads under flow.

3.
Nat Commun ; 12(1): 5552, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548486

##### RESUMO

Sepsis is a life-threatening condition caused by the extreme release of inflammatory mediators into the blood in response to infection (e.g., bacterial infection, COVID-19), resulting in the dysfunction of multiple organs. Currently, there is no direct treatment for sepsis. Here we report an abiotic hydrogel nanoparticle (HNP) as a potential therapeutic agent for late-stage sepsis. The HNP captures and neutralizes all variants of histones, a major inflammatory mediator released during sepsis. The highly optimized HNP has high capacity and long-term circulation capability for the selective sequestration and neutralization of histones. Intravenous injection of the HNP protects mice against a lethal dose of histones through the inhibition of platelet aggregation and migration into the lungs. In vivo administration in murine sepsis model mice results in near complete survival. These results establish the potential for synthetic, nonbiological polymer hydrogel sequestrants as a new intervention strategy for sepsis therapy and adds to our understanding of the importance of histones to this condition.

##### Assuntos
Hidrogéis/uso terapêutico , Nanopartículas/uso terapêutico , Sepse/tratamento farmacológico , Animais , Plaquetas/efeitos dos fármacos , Adesão Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Histonas/antagonistas & inibidores , Histonas/metabolismo , Histonas/toxicidade , Hidrogéis/química , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Nanopartículas/química , Nanopartículas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Ligação Proteica , Sepse/mortalidade , Taxa de Sobrevida
4.
Lab Chip ; 21(19): 3793-3803, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34581379

##### RESUMO

Single-cell analysis has become one of the main cornerstones of biotechnology, inspiring the advent of various microfluidic compartments for cell cultivation such as microwells, microtrappers, microcapillaries, and droplets. A fundamental assumption for using such microfluidic compartments is that unintended stress or harm to cells derived from the microenvironments is insignificant, which is a crucial condition for carrying out unbiased single-cell studies. Despite the significance of this assumption, simple viability or growth tests have overwhelmingly been the assay of choice for evaluating culture conditions while empirical studies on the sub-lethal effect on cellular functions have been insufficient in many cases. In this work, we assessed the effect of culturing cells in droplets on the cellular function using yeast morphology as an indicator. Quantitative morphological analysis using CalMorph, an image-analysis program, demonstrated that cells cultured in flasks, large droplets, and small droplets significantly differed morphologically. From these differences, we identified that the cell cycle was delayed in droplets during the G1 phase and during the process of bud growth likely due to the checkpoint mechanism and impaired mitochondrial function, respectively. Furthermore, comparing small and large droplets, cells cultured in large droplets were morphologically more similar to those cultured in a flask, highlighting the advantage of increasing the droplet size. These results highlight a potential source of bias in cell analysis using droplets and reinforce the significance of assessing culture conditions of microfluidic cultivation methods for specific study cases.

##### Assuntos
Saccharomyces cerevisiae , Análise de Célula Única , Biotecnologia , Técnicas de Cultura de Células , Microfluídica
5.
J Phys Chem Lett ; 12(32): 7859-7865, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34382803

##### RESUMO

Fluorescence-encoded vibrational spectroscopy has become increasingly more popular by virtue of its high chemical specificity and sensitivity. However, current fluorescence-encoded vibrational spectroscopy methods lack sensitivity in the low-frequency region, which if addressed could further enhance their capabilities. Here, we present a method for highly sensitive low-frequency fluorescence-encoded vibrational spectroscopy, termed fluorescence-encoded time-domain coherent Raman spectroscopy (FLETCHERS). By first exciting molecules into vibrationally excited states and then promoting the vibrating molecules to electronic states at varying times, the molecular vibrations can be encoded onto the emitted time-domain fluorescence intensity. We demonstrate the sensitive low-frequency detection capability of FLETCHERS by measuring vibrational spectra in the lower fingerprint region of rhodamine 800 solutions as dilute as 250 nM, which is â¼1000 times more sensitive than conventional vibrational spectroscopy. These results, along with further improvement of the method, open up the prospect of performing single-molecule vibrational spectroscopy in the low-frequency region.

##### Assuntos
Corantes Fluorescentes/química , Rodaminas/química , Análise Espectral Raman/métodos , Fluorescência , Limite de Detecção , Estudo de Prova de Conceito , Espectrometria de Fluorescência , Vibração
6.
Nat Commun ; 12(1): 3062, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031409

##### RESUMO

Raman optical activity (ROA) is effective for studying the conformational structure and behavior of chiral molecules in aqueous solutions and is advantageous over X-ray crystallography and nuclear magnetic resonance spectroscopy in sample preparation and cost performance. However, ROA signals are inherently minuscule; 3-5 orders of magnitude weaker than spontaneous Raman scattering due to the weak chiral light-matter interaction. Localized surface plasmon resonance on metallic nanoparticles has been employed to enhance ROA signals, but suffers from detrimental spectral artifacts due to its photothermal heat generation and inability to efficiently transfer and enhance optical chirality from the far field to the near field. Here we demonstrate all-dielectric chiral-field-enhanced ROA by devising a silicon nanodisk array and exploiting its dark mode to overcome these limitations. Specifically, we use it with pairs of chemical and biological enantiomers to show >100x enhanced chiral light-molecule interaction with negligible artifacts for ROA measurements.

7.
Trends Biotechnol ; 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33895013

##### RESUMO

The biophysical properties of cells reflect their identities, underpin their homeostatic state in health, and define the pathogenesis of disease. Recent leapfrogging advances in biophysical cytometry now give access to this information, which is obscured in molecular assays, with a discriminative power that was once inconceivable. However, biophysical cytometry should go 'deeper' in terms of exploiting the information-rich cellular biophysical content, generating a molecular knowledge base of cellular biophysical properties, and standardizing the protocols for wider dissemination. Overcoming these barriers, which requires concurrent innovations in microfluidics, optical imaging, and computer vision, could unleash the enormous potential of biophysical cytometry not only for gaining a new mechanistic understanding of biological systems but also for identifying new cost-effective biomarkers of disease.

8.
Environ Sci Technol ; 55(12): 7880-7889, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33913704

##### RESUMO

In the past few decades, microalgae-based bioremediation methods for treating heavy metal (HM)-polluted wastewater have attracted much attention by virtue of their environment friendliness, cost efficiency, and sustainability. However, their HM removal efficiency is far from practical use. Directed evolution is expected to be effective for developing microalgae with a much higher HM removal efficiency, but there is no non-invasive or label-free indicator to identify them. Here, we present an intelligent cellular morphological indicator for identifying the HM removal efficiency of Euglena gracilis in a non-invasive and label-free manner. Specifically, we show a strong monotonic correlation (Spearman's ρ = -0.82, P = 2.1 × 10-5) between a morphological meta-feature recognized via our machine learning algorithms and the Cu2+ removal efficiency of 19 E. gracilis clones. Our findings firmly suggest that the morphology of E. gracilis cells can serve as an effective HM removal efficiency indicator and hence have great potential, when combined with a high-throughput image-activated cell sorter, for directed-evolution-based development of E. gracilis with an extremely high HM removal efficiency for practical wastewater treatment worldwide.

##### Assuntos
Euglena gracilis , Metais Pesados , Microalgas , Biodegradação Ambiental , Citometria de Fluxo
9.
Acc Chem Res ; 54(9): 2132-2143, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33788539

##### RESUMO

Flow cytometry is a powerful tool with applications in diverse fields such as microbiology, immunology, virology, cancer biology, stem cell biology, and metabolic engineering. It rapidly counts and characterizes large heterogeneous populations of cells in suspension (e.g., blood cells, stem cells, cancer cells, and microorganisms) and dissociated solid tissues (e.g., lymph nodes, spleen, and solid tumors) with typical throughputs of 1,000-100,000 events per second (eps). By measuring cell size, cell granularity, and the expression of cell surface and intracellular molecules, it provides systematic insights into biological processes. Flow cytometers may also include cell sorting capabilities to enable subsequent additional analysis of the sorted sample (e.g., electron microscopy and DNA/RNA sequencing), cloning, and directed evolution. Unfortunately, traditional flow cytometry has several critical limitations as it mainly relies on fluorescent labeling for cellular phenotyping, which is an indirect measure of intracellular molecules and surface antigens. Furthermore, it often requires time-consuming preparation protocols and is incompatible with cell therapy. To overcome these difficulties, a different type of flow cytometry based on direct measurements of intracellular molecules by Raman spectroscopy, or "Raman flow cytometry" for short, has emerged. Raman flow cytometry obtains a chemical fingerprint of the cell in a nondestructive manner, allowing for single-cell metabolic phenotyping. However, its slow signal acquisition due to the weak light-molecule interaction of spontaneous Raman scattering prevents the throughput necessary to interrogate large cell populations in reasonable time frames, resulting in throughputs of about 1 eps. The remedy to this throughput limit lies in coherent Raman scattering methods such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS), which offer a significantly enhanced light-sample interaction and hence enable high-throughput Raman flow cytometry, Raman imaging flow cytometry, and even Raman image-activated cell sorting (RIACS). In this Account, we outline recent advances, technical challenges, and emerging opportunities of coherent Raman flow cytometry. First, we review the principles of various types of SRS and CARS and introduce several techniques of coherent Raman flow cytometry such as CARS, multiplex CARS, Fourier-transform CARS, SRS, SRS imaging flow cytometry, and RIACS. Next, we discuss a unique set of applications enabled by coherent Raman flow cytometry, from microbiology and lipid biology to cancer detection and cell therapy. Finally, we describe future opportunities and challenges of coherent Raman flow cytometry including increasing sensitivity and throughput, integration with droplet microfluidics, utilizing machine learning techniques, or achieving in vivo flow cytometry. This Account summarizes the growing field of high-throughput Raman flow cytometry and the bright future it can bring.

##### Assuntos
Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Humanos , Análise Espectral Raman
10.
Cytometry A ; 99(2): 127-128, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33522092
11.
Cell Res ; 31(7): 758-772, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33469157

##### RESUMO

Ca2+ channels are essential to cell birth, life, and death. They can be externally activated by optogenetic tools, but this requires robust introduction of exogenous optogenetic genes for expression of photosensitive proteins in biological systems. Here we present femtoSOC, a method for direct control of Ca2+ channels solely by ultrafast laser without the need for optogenetic tools or any other exogenous reagents. Specifically, by focusing and scanning wavelength-tuned low-power femtosecond laser pulses on the plasma membrane for multiphoton excitation, we directly induced Ca2+ influx in cultured cells. Mechanistic study reveals that photoexcited flavins covalently bind cysteine residues in Orai1 via thioether bonds, which facilitates Orai1 polymerization to form store-operated calcium channels (SOCs) independently of STIM1, a protein generally participating in SOC formation, enabling all-optical activation of Ca2+ influx and downstream signaling pathways. Moreover, we used femtoSOC to demonstrate direct neural activation both in brain slices in vitro and in intact brains of living mice in vivo in a spatiotemporal-specific manner, indicating potential utility of femtoSOC.

12.
Trends Biotechnol ; 39(10): 978-989, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33509656

##### RESUMO

Technological advances in image-based platelet analysis or platelet morphometry are critical for a better understanding of the structure and function of platelets in biological research as well as for the development of better clinical strategies in medical practice. Recently, the advent of high-throughput optical imaging and deep learning has boosted platelet morphometry to the next level by providing a new set of capabilities beyond what is achievable with traditional platelet morphometry, shedding light on the unexplored domain of platelet analysis. This Opinion article introduces emerging opportunities in 'intelligent' platelet morphometry, which are expected to pave the way for a new class of diagnostics, pharmacometrics, and therapeutics.

13.
Opt Express ; 28(20): 29272-29284, 2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-33114830

##### RESUMO

Optical time-stretch (OTS) imaging is effective for observing ultra-fast dynamic events in real time by virtue of its capability of acquiring images with high spatial resolution at high speed. In different implementations of OTS imaging, different configurations of its signal detection, i.e. fiber-coupled and free-space detection schemes, are employed. In this research, we quantitatively analyze and compare the two detection configurations of OTS imaging in terms of sensitivity and image quality with the USAF-1951 resolution chart and diamond films, respectively, providing a valuable guidance for the system design of OTS imaging in diverse fields.

14.
Opt Express ; 28(21): 31914-31922, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33115155

##### RESUMO

We present sequentially timed all-optical mapping photography (STAMP) with a slicing mirror in a branched 4f system for an increased number of frames without sacrificing pixel resolution. The branched 4f system spectrally separates the laser light path into multiple paths by the slicing mirror placed in the Fourier plane. Fabricated by an ultra-precision end milling process, the slicing mirror has 18 mirror facets of differing mirror angles. We used the boosted STAMP to observe dynamics of laser ablation with two image sensors which captured 18 subsequent frames at a frame rate of 126 billion frames per second, demonstrating this technique's potential for imaging unexplored ultrafast non-repetitive phenomena.

15.
Nat Commun ; 11(1): 4772, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973145

##### RESUMO

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for vibrational spectroscopy as it provides several orders of magnitude higher sensitivity than inherently weak spontaneous Raman scattering by exciting localized surface plasmon resonance (LSPR) on metal substrates. However, SERS can be unreliable for biomedical use since it sacrifices reproducibility, uniformity, biocompatibility, and durability due to its strong dependence on "hot spots", large photothermal heat generation, and easy oxidization. Here, we demonstrate the design, fabrication, and use of a metal-free (i.e., LSPR-free), topologically tailored nanostructure composed of porous carbon nanowires in an array as a SERS substrate to overcome all these problems. Specifically, it offers not only high signal enhancement (~106) due to its strong broadband charge-transfer resonance, but also extraordinarily high reproducibility due to the absence of hot spots, high durability due to no oxidization, and high compatibility to biomolecules due to its fluorescence quenching capability.

##### Assuntos
Carbono/química , Nanofios/química , Análise Espectral Raman/métodos , Fluorescência , Porosidade , Reprodutibilidade dos Testes , Ressonância de Plasmônio de Superfície/métodos , Propriedades de Superfície
16.
Lab Chip ; 20(17): 3074-3090, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32644061

##### RESUMO

Artificial intelligence (AI) has dramatically changed the landscape of science, industry, defence, and medicine in the last several years. Supported by considerably enhanced computational power and cloud storage, the field of AI has shifted from mostly theoretical studies in the discipline of computer science to diverse real-life applications such as drug design, material discovery, speech recognition, self-driving cars, advertising, finance, medical imaging, and astronomical observation, where AI-produced outcomes have been proven to be comparable or even superior to the performance of human experts. In these applications, what is essentially important for the development of AI is the data needed for machine learning. Despite its prominent importance, the very first process of the AI development, namely data collection and data preparation, is typically the most laborious task and is often a limiting factor of constructing functional AI algorithms. Lab-on-a-chip technology, in particular microfluidics, is a powerful platform for both the construction and implementation of AI in a large-scale, cost-effective, high-throughput, automated, and multiplexed manner, thereby overcoming the above bottleneck. On this platform, high-throughput imaging is a critical tool as it can generate high-content information (e.g., size, shape, structure, composition, interaction) of objects on a large scale. High-throughput imaging can also be paired with sorting and DNA/RNA sequencing to conduct a massive survey of phenotype-genotype relations whose data is too complex to analyze with traditional computational tools, but is analyzable with the power of AI. In addition to its function as a data provider, lab-on-a-chip technology can also be employed to implement the developed AI for accurate identification, characterization, classification, and prediction of objects in mixed, heterogeneous, or unknown samples. In this review article, motivated by the excellent synergy between AI and lab-on-a-chip technology, we outline fundamental elements, recent advances, future challenges, and emerging opportunities of AI with lab-on-a-chip technology or "AI on a chip" for short.

##### Assuntos
Inteligência Artificial , Dispositivos Lab-On-A-Chip , Algoritmos , Humanos , Aprendizado de Máquina , Modelos Teóricos
17.
Nat Commun ; 11(1): 3452, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651381

##### RESUMO

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

##### Assuntos
Separação Celular/métodos , Análise Espectral Raman/métodos , Animais , Humanos
18.
Elife ; 92020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32393438

##### RESUMO

Platelets are anucleate cells in blood whose principal function is to stop bleeding by forming aggregates for hemostatic reactions. In addition to their participation in physiological hemostasis, platelet aggregates are also involved in pathological thrombosis and play an important role in inflammation, atherosclerosis, and cancer metastasis. The aggregation of platelets is elicited by various agonists, but these platelet aggregates have long been considered indistinguishable and impossible to classify. Here we present an intelligent method for classifying them by agonist type. It is based on a convolutional neural network trained by high-throughput imaging flow cytometry of blood cells to identify and differentiate subtle yet appreciable morphological features of platelet aggregates activated by different types of agonists. The method is a powerful tool for studying the underlying mechanism of platelet aggregation and is expected to open a window on an entirely new class of clinical diagnostics, pharmacometrics, and therapeutics.

##### Assuntos
Redes Neurais de Computação , Agregação Plaquetária , Citometria de Fluxo , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Ativação Plaquetária , Trombose/classificação
19.
Lab Chip ; 20(13): 2263-2273, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32459276

##### RESUMO

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of â¼2000 events per second and a high sensitivity of â¼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

##### Assuntos
Redes Neurais de Computação , Software , Algoritmos , Separação Celular , Citometria de Fluxo
20.
Opt Lett ; 45(8): 2339-2342, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32287228

##### RESUMO

We propose and experimentally demonstrate high-speed single-pixel imaging by integrating frequency-division multiplexing and time-division multiplexing (techniques used widely in telecommunications) and applying the combined technique, namely, frequency-time-division multiplexing (FTDM), to optical imaging. Specifically, FTDM single-pixel imaging uses an array of broadband, spatially distributed, dual-frequency combs (i.e., spatial dual combs) for multidimensional illumination and detects an image-encoded time-domain signal with a single-pixel photodetector in a FTDM manner. As a proof-of-principle demonstration, we use the method to show ultrafast two-color (bright-field and fluorescence) single-pixel microscopy of breast cancer cells at a high frame rate of 32,000 fps and ultrafast image velocimetry of fluorescent particles flowing at a high speed of ${ \gt }{2}\;{\rm m/s}$>2m/s.

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