- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
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
-
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
-
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
-
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
-
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
-
A novel intracellular signal amplification strategy for the quantification of ATP in single cells by microchip electrophoresis with laser induced fluorescence detection
摘要： An intracellular signal amplification strategy was developed for quantification of ATP in single cells by microchip electrophoresis with laser induced fluorescence detection. By using the method proposed, intracellular ATP levels in single Hela, HepG2 and HL-7702 cell were found in the range of 30～150, 30~140, and 19～120 fmol/cell, respectively.
关键词： laser induced fluorescence,ATP,microchip electrophoresis,single-cell analysis,signal amplification
更新于2025-09-23 15:21:01
-
Metabolic mapping with plasmonic nanoparticle assemblies
摘要： A rapid and simple methodology for the biomolecular analysis of single cells and microenvironments via a stick-and-peel plasmonic sensing platform is reported. Substrate-bound assemblies of plasmonic gold nanoparticles linked by reconfigurable oligonucleotides undergo disassembly upon target binding. Changes in the light scattering intensity of thousands of discrete nanoparticle assemblies are extrapolated concomitantly to yield the mapping of local target concentrations. The methodology is completely free of labelling, purification and separation steps. We quantified the intracellular ATP levels for two ovarian cancer cell lines to elucidate the differences and cellular distribution, and demonstrated the potential of the stick-and-peel platform for mapping the microenvironment of a 2D heterogeneous surface. The portable and economical analytical platform may broaden the affordability and applicability of single-cell based analyses and enable new opportunities in clinical care such as on-site molecular pathology.
关键词： stick-and-peel platform,plasmonic nanoparticle assemblies,ATP detection,single-cell analysis,molecular pathology
更新于2025-09-23 15:19:57
-
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
-
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
-
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