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Automated nanoplasmonic analysis of spherical nucleic acids clusters in single cells
摘要： Spherical nucleic acids (SNAs) have been extensively used in the field of biosensing, drug delivery and theranostics. Precise engineering of SNAs and their clinical application require better understanding of their cellular internalization process. We demonstrate a colorimetry-based algorithm (CBA) that can analyze the aggregation states of SNAs clusters based on the changes of plasmonic colors of SNAs. The dark field microscopy (DFM) images of cytoplasmic region of single cells are imported as raw data. All the image spots are analyzed in the interference reduction process, and the clustering states of target image spots are assigned based on the distribution of coordinates of all the pixels in the CIE map. This method provides faster analysis on clustering states of extracellular and intracellular SNAs with good accuracy. Moreover, the clustering states of SNAs in 20 single cells (generally more than 1000) can be efficiently distinguished within 200 seconds. Therefore, our method provides an automatic, quantitative, objective and repeatable way to analyze SNAs aggregations, and shows good application potential in robust and quantitative nanoplasmonic analysis in single cells.
关键词： Single-cell analysis,Plasmonic analysis,Single-particle analysis,Spherical nucleic acids,Clustering states
更新于2025-09-12 10:27:22
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Rapid Assessment of Water Toxicity by Plasmonic Nanomechanical Sensing
摘要： The ability to rapidly and accurately detect water toxicity is crucial for monitoring water quality and assessing toxic risk, but such detection remains a great challenge. Here, we present a plasmonic nanomechanical sensing (PNMS) system for the rapid assessment of water toxicity. This technique is based on the plasmonic sensing of the nanomechanical movement of single bacterial cells, which could be inhibited upon exposure to potential toxicants. By correlating the amplitude of nanomechanical movement with bacterial activity, we detected a variety of toxic substances in water. The direct readout of bacterial activity via PNMS allowed for a high sensitivity to toxicants in water, thereby enabling us to evaluate the acute toxicological effect of chemical compounds rapidly. The PNMS method is promising for online alerts of water quality safety and for assessing chemical hazards. We anticipate that PNMS is also suitable for a wide range of other applications, including bacterial detection and high-throughput screening of anti-bacterial materials.
关键词： water toxicity,nanomechanical movement,high-throughput,detection,Single cell
更新于2025-09-12 10:27:22
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Immune cell type, cell activation, and single cell heterogeneity revealed by label-free optical methods
摘要： Measurement techniques that allow the global analysis of cellular responses while retaining single-cell sensitivity are increasingly needed in order to understand complex and dynamic biological processes. in this context, compromises between sensitivity, degree of multiplexing, throughput, and invasiveness are often unavoidable. We present here a noninvasive optical approach that can retrieve quantitative biomarkers of both morphological and molecular phenotypes of individual cells, based on a combination of quantitative phase imaging and Raman spectroscopy measurements. We then develop generalized statistical tools to assess the influence of both controlled (cell sub-populations, immune stimulation) and uncontrolled (culturing conditions, animal variations, etc.) experimental parameters on the label-free biomarkers. These indicators can detect different macrophage cell sub-populations originating from different progenitors as well as their activation state, and how these changes are related to specific differences in morphology and molecular content. The molecular indicators also display further sensitivity that allow identification of other experimental conditions, such as differences between cells originating from different animals, allowing the detection of outlier behaviour from given cell sub-populations.
关键词： single cell heterogeneity,label-free optical methods,cell activation,immune cell type
更新于2025-09-11 14:15:04
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Plasmonic droplet screen for single-cell secretion analysis
摘要： Single-cell secretion analysis technologies are needed to elucidate the heterogeneity of cellular functionalities. Although ligand binding assays in microwells provide a promising approach for measuring single-cell secretions, their throughput is limited. Recently, droplet assays have been developed for high-throughput single-cell screening. However, because washing steps are difficult to perform with droplets, there are still challenges in measuring secretions using droplet assays. In this study, a plasmonic droplet screen approach is developed for one-step washing-free multiplex detection of single-cell secretions. Individual cells are encapsulated with antibody-conjugated gold nanorods (AuNRs) in droplets to evaluate their secretion levels. The shift in the plasmon resonance peak reflects the amount of secreted protein without needing additional indicator and washing steps. The plasmonic signals from a continuous flow of single-cell droplets are collected by dark-field spectroscopy (～100–150 cells min?1). This platform is tested by screening interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) secreted from suspended leukemia cells and adherent breast cancer cells. Overall, this novel strategy shows the potential and flexibility of high-efficiency multiplex single-cell secretion analysis.
关键词： Multiplex detection,Platform engineering,Immunoassay,Surface plasmon resonance,Single cell analysis
更新于2025-09-11 14:15:04
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Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ
摘要： RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ’s compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.
关键词： laser-capture microdissection,single-cell gene expression,RNA-seq,tumor microenvironment,FFPE tissue,Smart-3SEQ
更新于2025-09-11 14:15:04
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Confined electrochemiluminescence in vertically ordered silica mesochannels for the imaging of hydrogen peroxide released from single cells
摘要： Here, the confined electrochemiluminescence (ECL) of luminol and hydrogen peroxide is achieved in vertically ordered silica mesochannels (SMCs) and applied to map the efflux of hydrogen peroxide from single living cells. The vertical alignment of SMCs on indium tin oxide (ITO) slides restricts the lateral diffusion of species generated in the ECL process. Accordingly, the cross-talk of luminescence intensity from nearby microregions is minimized for higher-spatial-resolution ECL imaging. The characterization of luminol ECL on these SMC-coated slides yields a detection range of hydrogen peroxide between 5 μM and 1 mM, providing high sensitivity in imaging the efflux of hydrogen peroxide from cells. Compared with the luminescence from individual cells on bare ITO slides, ~ 4-fold intensity is observed for SMC-coated ITO, suggesting a better electrode surface for single-cell ECL imaging.
关键词： single-cell imaging,hydrogen peroxide,silica mesochannels,confined electrochemiluminescence,luminol
更新于2025-09-10 09:29:36
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Nanoscale tweezers for single-cell biopsies
摘要： Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional ‘snapshot’ of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10–20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.
关键词： single-cell biopsies,nanotweezers,proteins,DNA,mitochondrion,dielectrophoretic trapping
更新于2025-09-10 09:29:36
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BCAbox Algorithm Expands Capabilities of Raman Microscope for Single Organelles Assessment
摘要： Raman microspectroscopy is a rapidly developing technique, which has an unparalleled potential for in situ proteomics, lipidomics, and metabolomics, due to its remarkable capability to analyze the molecular composition of live cells and single cellular organelles. However, the scope of Raman spectroscopy for bio-applications is limited by a lack of software tools for express-analysis of biomolecular composition based on Raman spectra. In this study, we have developed the first software toolbox for immediate analysis of intracellular Raman spectra using a powerful biomolecular component analysis (BCA) algorithm. Our software could be easily integrated with commercial Raman spectroscopy instrumentation, and serve for precise analysis of molecular content in major cellular organelles, including nucleoli, endoplasmic reticulum, Golgi apparatus, and mitochondria of either live or fixed cells. The proposed software may be applied in broad directions of cell science, and serve for further advancement and standardization of Raman spectroscopy.
关键词： single cell assay,biomolecular analysis,Raman spectrometry,microscopy
更新于2025-09-09 09:28:46
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Dynamic Microfluidic Cytometry for Single-Cell Cellomics: High-throughput Probing Single-Cell-Resolution Signaling
摘要： Cell signaling is a fast dynamic and complex process, which controls a variety of critical physiological functions. Methods to investigate such dynamic information, however, suffer from limited throughput in single cells level and a lack of precise fluid manipulation. Herein, we present a new strategy, termed dynamic microfluidic cytometry (DMC), for high throughput probing GPCR signaling in single cells resolution (single-cell cellomics analysis) by creatively applied cyclical cell trapping, stimulating and releasing automatically. Dose-response curves and EC50 values for HeLa cells treated with adenosine triphosphate (ATP), histamine (HA) and acetylcholine chloride (ACH) were successfully obtained in single-cells level. High throughput single-cells dynamic signaling were further implemented by sequential or simultaneous stimulation, which revealed that different mechanisms were working in triggering intracellular calcium releasing. In addition, simultaneous stimulating to two different types of cells, HeLa and NIH-3T3 cells, was also successfully realized, which was crucial for online comparison dynamic signaling of different types of cells. We believe that the proposed DMC provides a versatile means for high throughput probing single cell dynamic signaling, which is potentially useful in chemical biology, cell biology and pharmacology.
关键词： dynamic signaling,cytometry,microfluidics,single-cell cellomics
更新于2025-09-04 15:30:14
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Quantifying single-cell secretion in real time using resonant hyperspectral imaging
摘要： Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.
关键词： single-cell analysis,photonic biosensing,photonic crystal,label-free
更新于2025-09-04 15:30:14