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- 实验方案
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Surface Coatings Modulate the Differences in the Adhesion Forces of Eukaryotic and Prokaryotic Cells as Detected by Single Cell Force Microscopy
摘要： Single cell force microscopy was used to investigate the maximum detachment force (MDF) of primary neuronal mouse cells (PNCs), osteoblastic cells (MC3T3), and prokaryotic cells (Staphylococcus capitis subsp. capitis) from different surfaces after contact times of 1 to 5 seconds. Positively charged silicon nitride surfaces were coated with positively charged polyethyleneimine (PEI) or poly-D-lysine. Laminin was used as the second coating. PEI induced MDFs of the order of 5 to 20 nN, slightly higher than silicon nitride did. Lower MDFs (1 to 5 nN) were detected on PEI/laminin with the lowest on PDL/laminin. To abstract from the individual cell properties, such as size, and to obtain cell type-specific MDFs, the MDFs of each cell on the different coatings were normalized to the silicon nitride reference for the longest contact time. The differences in MDF between prokaryotic and eukaryotic cells were generally of similar dimensions, except on PDL/laminin, which discriminated against the prokaryotic cells. We explain the lower MDFs on laminin by the spatial prevention of the electrostatic cell adhesion to the underlying polymers. However, PEI can form long flexible loops protruding from the surface-bound layer that may span the laminin layer and easily bind to cellular surfaces and the small prokaryotic cells. This was reflected in increased MDFs after two-second contact times on silicon nitride, whereas the two-second values were already observed after one second on PEI or PEI/laminin. We assume that the electrostatic charge interaction with the PEI loops is more important for the initial adhesion of the smaller prokaryotic cells than for eukaryotic cells.
关键词： prokaryotic cells,poly-D-lysine,silicon nitride,laminin,cell adhesion,single cell force microscopy,surface coatings,polyethyleneimine,eukaryotic cells,maximum detachment force
更新于2025-11-21 11:24:58
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Single-cell redox states analyzed by fluorescence lifetime metrics and tryptophan FRET interaction with NAD(P)H
摘要： Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens.
关键词： Fluorescence Lifetime Imaging Microscopy (FLIM),single-cell analysis,NADPH/NADH ratio,NAD(P)H,redox,FAD,fluorescence lifetime redox ratio (FLIRR),NAD(P)H-a2%
更新于2025-11-21 11:24:58
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Graphene quantum dots enhanced ToF-SIMS for single-cell imaging
摘要： Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has shown promising applications in single-cell analysis owing to its high spatial resolution molecular imaging capability. One of the main drawbacks hindering progress in this field is the relatively low ionization efficiency for biological systems. The complex chemical micro-environment in single cells typically causes severe matrix effects, leading to significant signal suppression of biomolecules. In this work, we investigated the signal enhancement effect of graphene quantum dots (GE QDs) in ToF-SIMS analysis. A × 160 magnification of ToF-SIMS signal for amiodarone casted on glass slide was observed by adding amino-functionalized GE QDs (amino-GE QDs), which was significantly higher than adding previously reported signal enhancement materials and hydroxyl group-functionalized GE QDs (hydroxyl-GE QDs). A possible mechanism for GE QD-induced signal enhancement was proposed. Further, effects of amino-GE QDs and hydroxyl-GE QDs on amiodarone-treated breast cancer cells were compared. A significant signal improvement for lipids and amiodarone was achieved using both types of GE QDs, especially for amino-GE QDs. In addition, ToF-SIMS chemical mapping of single cells with better quality was obtained after signal enhancement. Our strategy for effective ToF-SIMS signal enhancement holds great potential for further investigation of drug metabolism pathways and the interactions between the cell and micro-environment.
关键词： Signal enhancement,Single-cell analysis,Graphene quantum dots,Time-of-flight secondary ion mass spectrometry
更新于2025-11-14 15:32:45
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Dynamic fluorescent imaging analysis of mitochondrial redox in single cells with a microfluidic device
摘要： The redox balance in celluar mitochondria is closely related to the physiological and pathological processes of the body. When exposed to external stimuli, the redox state in cells changes dynamically, and presents cell heterogeneity, which creates a need for techniques that can make dynamic and reversible visual analysis of redox in mitochondria at single-cell level. Here we describe a method for single-cell redox analysis based on a microfluidic device combing with a reversible fluorescent probe (Cy-O-ebselen) , that enables online culture, labelling and dynamic fluorescent imaging analysis of mitochondrial redox (H2O2/GSH) change. Using this method, we further explored the dynamic changes of mitochondrial redox state after thermal stimulation or combined thermal-drug stimulation, and analyzed the heterogeneous response of cells to external stimuli at the single cell level.
关键词： single cell,fluorescence,microfluidic,mitochondrial redox
更新于2025-09-23 15:23:52
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Ultrasensitive and simultaneous detection of two cytokines secreted by single cell in microfluidic droplets via magnetic-field amplified SERS
摘要： A surface-enhanced Raman scattering (SERS)-microfluidic droplet platform for the rapid, ultrasensitive and simultaneous detection of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) secreted by a single cell is presented. The high throughput water-in-oil droplets containing individual cell along with four kinds of immune-particles (antibody-conjugated silver nanoparticles or magnetic beads, AgNPs@Ab1 and MNs@Ab2) in each were achieved by a cross-typed microfluidic chip, and then they were captured by a collection channel array for SERS measurements. In the appearance of cytokines secreted by one cell, AgNPs@Ab1 can be linked onto the surface of MNs@Ab2 through the immune-recognition to form an immune-sandwich, which makes the 'turn on' SERS signal of the Raman reporters previously laid on the surface of MNs due to the adjacent AgNPs. Furthermore, the second SERS signal amplification is from the magnetic field-induced spontaneous collection effect, which brings 75 times enhancement for SERS signal. Additionally, the encapsulation of cytokines in an isolated droplet permits an accumulation effect of targets with time. Owing to the dual signal enhancement and the accumulation effect, such few cytokines secreted by single cell become detectable and a limit of detection is achieved as 1.0 fg/mL in one droplet. By using this ultrasensitive SERS-microdroplet method, the VEGF and IL-8 secretions from several cells in one droplet were explored and the data show that the cell–cell interactions may promote angiogenesis of cancer cells through the up-regulation of VEGF and IL-8.
关键词： surface-enhanced Raman scattering,VEGF,magnetic field amplification,single-cell analysis,IL-8,cytokines,microfluidic droplets
更新于2025-09-23 15:23:52
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Chemical and topographical single-cell imaging by near-field desorption mass spectrometry
摘要： Simultaneous acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge for current techniques due to limited lateral resolutions and detection sensitivities. Here we report the development of near-field desorption mass spectrometry for co-registered chemical and topographical imaging, thereby bridging the gap between laser-based MS methods and multimodal single-cell imaging. Using this integrated platform, imaging resolution of 250 nm and 3D topographically reconstructed chemical single-cell imaging were achieved. This technique offers more in-depth cellular information than is currently possible with micrometre-range laser-based MS imaging methods. Considering the simplicity and compact size of the near-field device, this technique can be introduced to matrix-assisted laser desorption ionization MS, expanding the multimodal abilities of MS at nanoscale resolutions across wide disciplines.
关键词： single-cell analysis,analytical methods,multimodal imaging,mass spectrometry,near-field desorption
更新于2025-09-23 15:23:52
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Determination of formaldehyde in single cell by capillary electrophoresis with LIF detection
摘要： As a small molecule gas, formaldehyde (FA) is the simplest carbonyl active material and plays an important role in the carbon cycle of metabolism. However, due to the volatile nature of the gas, it is difficult to accurately quantify its content, which limits the study of the mechanism of action in life activities. Thus, an efficient approach to quantitative detection of FA in cells especially in single cell is urgent needed. Nevertheless, no method for quantifying FA in single cell has been reported to date. In this work, a fluorescent probe N-propyl-4-hydrazino-naphthalimide (NPHNA), which has highly desirable attributes and has been applied to living biological samples, was chosen as labeling reagent to detect endogenous FA at single cell level. After optimization of separation conditions, fast baseline separation of the FA derivative N-propyl-4-hydrazone-naphthalimide product (NPHNA-FA) and NPHNA was achieved in about 5 min by capillary electrophoresis with laser-induced fluorescence detection. The detection limit for FA was 5 amol (signal-to-noise ratio of 3). The developed method was validated by the measurements of intracellular levels of FA in single cell.
关键词： Capillary electrophoresis,Endogenous formaldehyde levels,Single cell
更新于2025-09-23 15:22:29
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A single-cell-gap transflective liquid crystal display with a vertically aligned cell
摘要： A single-cell-gap transflective liquid crystal display with a vertically aligned cell using square ring electrode is demonstrated. The top substrate has a top planar common electrode, a square ring pixel electrode is coated on the bottom substrate, while a bumpy reflector is coated under the bottom substrate. In this device, the planar common electrode and square ring pixel electrode generate a strong longitudinal electric field in the transmissive region (T region) and a weak fringe field in the reflective region (R region). As result, the T and R regions accumulate the same optical phase retardation. The simulation results show that the display exhibits reasonably low operating voltage, high transmittance and well-matched voltage-dependent transmittance and reflectance curves. Besides, fabrication process of the transflective liquid crystal display is very simple.
关键词： square ring electrode,Vertically aligned cell,negative dielectric anisotropy,single-cell-gap,transflective liquid crystal display
更新于2025-09-23 15:21:21
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Light-microscopy methods in C. elegans research
摘要： Ever since Caenorhabditis elegans was introduced as a model system it has been tightly linked to microscopy, which has led to significant advances in understanding biology over the last decades. Developing new technologies therefore is an essential part in the endeavor to gain further mechanistic insights into developmental biology. This review will discuss state-of-the-art developments in quantitative light microscopy in the context of C. elegans research as well as the impact these technologies have on the field. We will highlight future developments that currently promise to revolutionize biological research by combining sequencing-based single-cell technologies with high-resolution quantitative imaging.
关键词： quantitative imaging,light microscopy,developmental biology,single-cell technologies,C. elegans
更新于2025-09-23 15:21:21
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Luminescent nanomaterials for droplet tracking in a microfluidic trapping array
摘要： The use of high-throughput multiplexed screening platforms has attracted significant interest in the field of on-site disease detection and diagnostics for their capability to simultaneously interrogate single-cell responses across different populations. However, many of the current approaches are limited by the spectral overlap between tracking materials (e.g., organic dyes) and commonly used fluorophores/biochemical stains, thus restraining their applications in multiplexed studies. This work demonstrates that the downconversion emission spectra offered by rare earth (RE)-doped β-hexagonal NaYF4 nanoparticles (NPs) can be exploited to address this spectral overlap issue. Compared to organic dyes and other tracking materials where the excitation and emission is separated by tens of nanometers, RE elements have a large gap between excitation and emission which results in their spectral independence from the organic dyes. As a proof of concept, two differently doped NaYF4 NPs (europium: Eu3+, and terbium: Tb3+) were employed on a fluorescent microscopy-based droplet microfluidic trapping array to test their feasibility as spectrally independent droplet trackers. The luminescence tracking properties of Eu3+-doped (red emission) and Tb3+-doped (green emission) NPs were successfully characterized by co-encapsulating with genetically modified cancer cell lines expressing green or red fluorescent proteins (GFP and RFP) in addition to a mixed population of live and dead cells stained with ethidium homodimer. Detailed quantification of the luminescent and fluorescent signals was performed to confirm no overlap between each of the NPs and between NPs and cells. Thus, the spectral independence of Eu3+-doped and Tb3+-doped NPs with each other and with common fluorophores highlights the potential application of this novel technique in multiplexed systems, where many such luminescent NPs (other doped and co-doped NPs) can be used to simultaneously track different input conditions on the same platform.
关键词： Rare earth elements,Single-cell analysis,Nanoparticles,Microfluidics,high-throughput screening
更新于2025-09-23 15:21:21