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Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation
摘要： Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (?: 80 μm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.
关键词： microdroplets,hydrogels,single cell analysis,stem cells,pluripotency
更新于2025-09-23 15:19:57
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An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages
摘要： Purpose To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. Methods The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. Results Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified Bfoamy^ lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. Conclusions This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.
关键词： lipid-laden foamy macrophages,intracellular drug bioaccumulation,single-cell chemical imaging,confocal Raman microscopy,pulmonary alveolar macrophages
更新于2025-09-23 15:19:57
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High-throughput label-free molecular fingerprinting flow cytometry
摘要： Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm?1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics of Haematococcus lacustris.
关键词： high-throughput,astaxanthin,label-free,single-cell analysis,microfluidics,flow cytometry,Raman spectroscopy,Haematococcus lacustris,coherent anti-Stokes Raman scattering
更新于2025-09-19 17:15:36
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Detection of Metabolic Changes Induced via Drug Treatments in Live Cancer Cells and Tissue Using Raman Imaging Microscopy
摘要： Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD+) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue.
关键词： NAD+ synthesis,tissue imaging,single cell imaging,microscopy,Raman spectrometry,fibrosarcoma IDH1
更新于2025-09-19 17:15:36
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Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer
摘要： Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.
关键词： single-cell RNA-Seq,metastasis,circulating tumor cells,GEMM,microfluidic
更新于2025-09-19 17:15:36
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Combination of an Artificial Intelligence Approach and Laser Tweezers Raman Spectroscopy for Microbial Identification
摘要： Raman spectroscopy is a non-destructive, label-free, highly-specific approach which provides the chemical information of materials. Thus, it is suitable to be used as an effective analytical tool to characterize biological samples. Here we introduce a novel method which uses artificial intelligence to analyze biological Raman spectra and identify the microbes at a single-cell level. The combination of a framework of convolutional neural network (ConvNet) and Raman spectroscopy allows the extraction of the Raman spectral features of a single microbial cell and then categorizes cells according to their spectral features. As the proof of concept, we measured Raman spectra of 14 microbial species at a single-cell level and constructed an optimal ConvNet model using the Raman data. The average accuracy of classification by ConvNet is 95.64±5.46%. Meanwhile, we introduced an occlusion-based Raman spectra feature extraction (ORSFE) to visualize the weights of Raman features for distinguishing different species.
关键词： convolutional neural network,single cell,Raman feature extraction,Raman spectroscopy
更新于2025-09-19 17:13:59
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Single-Cell Optogenetic Control of Calcium Signaling with a High-Density Micro-LED Array
摘要： Precise optogenetic control, ideally down to single cells in dense cell populations, is essential in understanding the heterogeneity of cell networks. Devices with such capability, if built in a chip scale, will advance optogenetic studies at cellular levels in a variety of experimental settings. Here we demonstrate optogenetic control of intracellular Ca2+ dynamics at the single cell level using a 16-μm pitched micro-LED array that features high brightness, small spot size, fast response, and low voltage operation. Individual LED pixels are able to reliably trigger intracellular Ca2+ transients, confirmed by fluorescence microscopy, control experiments, and cross-checked by two genetically-coded Ca2+ indicators. Importantly, our array can optogenetically address individual cells that are sub-10 μm apart in densely packed cell populations. These results suggest the possible use of the micro-LED array towards a lab-on-a-chip for single-cell optogenetics, which may allow for pharmaceutical screening and fundamental studies on a variety of cell networks.
关键词： optogenetic control,lab-on-a-chip,calcium signaling,micro-LED array,single-cell
更新于2025-09-19 17:13:59
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Correlated Secondary Ion Mass Spectrometry-Laser Scanning Confocal Microscopy Imaging for Single Cell-Principles and Applications
摘要： Secondary ion mass spectrometry (SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80?100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1–5 nm. However, owing to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still have great challenges. Optical microscopy, in particular laser scanning confocal microscopy (LSCM), has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.
关键词： Cell biology,Single cell imaging,Laser scanning confocal microscopy,Correlated secondary ion mass spectrometry and laser scanning confocal microscopy imaging,Secondary ion mass spectrometry,Review
更新于2025-09-16 10:30:52
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Laser cleavable probes for <i>in situ</i> multiplexed glycan detection by single cell mass spectrometry
摘要： Glycans binding on the cell surface through glycosylation play a key role in controlling various cellular processes, and glycan analysis at a single-cell level is necessary to study cellular heterogeneity and diagnose diseases in the early stage. Herein, we synthesized a series of laser cleavable probes, which could sensitively detect glycans on single cells and tissues by laser desorption ionization mass spectrometry (LDI-MS). This multiplexed and quantitative glycan detection was applied to evaluate the alterations of four types of glycans on breast cancer cells and drug-resistant cancer cells at a single-cell level, indicating that drug resistance may be related to the upregulation of glycan with a b-D-galactoside (Galb) group and Neu5Aca2-6Gal(NAc)-R. Moreover, the glycan spatial distribution in cancerous and paracancerous human tissues was also demonstrated by MS imaging, showing that glycans are overexpressed in cancerous tissues. Therefore, this single-cell MS approach exhibits promise for application in studying glycan functions which are essential for clinical biomarker discovery and diagnosis of related diseases.
关键词： mass spectrometry,breast cancer,drug resistance,laser cleavable probes,single-cell analysis,glycans
更新于2025-09-16 10:30:52
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Plasmonic Gold Templates Enhancing Single Cell Lipidomic Analysis of Microorganisms
摘要： Single cell lipid profiling is a powerful tool to connect membrane composition and its changes within individual cells to specific biochemical functions or stimuli, but current approaches are inadequate due to the complex nature of the cells and technical limitation in analysis. Herein we report a new method with plasmonic substrates capable of cell localization and enhanced lipid ionization through thin-gold-film MALDI-MS. We performed lipidomic profiling of algae single cells with a 120-well microarray and identified more than 50 lipids in C. reinhardtii without an extraction process. The substrate was used for probing toxicological effect of herbicide atrazine on the algae’s lipidome, demonstrating molecular changes in glycerol lipid profiles. Fast location of cells with metal-enhanced fluorescence (MEF) and subsequent precise and direct ionization of the LDI process contribute to the enhanced performance, allowing for assessment of lipid changes concurrent with atrazine affected populations. This method that combines microarrays, MEF and MALDI-MS presents an effective platform for lipidomic study of single cells and for environmental toxicity study with microorganisms.
关键词： plasmonic substrates,MALDI-MS,atrazine,environmental toxicity,lipidomic profiling,single cell lipid profiling
更新于2025-09-16 10:30:52