Position-referenced microscopy for live cell culture monitoring.
Ontology highlight
ABSTRACT:
SUBMITTER: Galeano Z JA
PROVIDER: S-EPMC3087587 | biostudies-other | 2011
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Growth rate is a widely studied parameter for various cell-based biological studies. Growth rates of cell populations can be monitored in chemostats and micro-chemostats, where nutrients are continuously replenished. Here, we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of (i) growth rates of cells by means of impedance measurements and of (ii) specific other cellular events by means of high-resolution optical or fluorescence microscopy. Yeast colonies were grown in a monolayer under culturing pads, which enabled high-resolution microscopy, as all cells were in the same focal plane. Upon cell growth and division, cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements. The impedance data could then be used to directly determine the growth rates of the cells in the culturing area. The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel. As a demonstration, we modulated the growth rates of engineered yeast strains using calcium. The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells.
| S-EPMC6220194 | biostudies-literature
Project description:We present here a label-free development based on preexisting Quantitative Phase Imaging (QPI) that allows non-invasive live monitoring of both individual cells and cell populations. Growth, death, effect of toxic compounds are quantified under visible light with a standard inverted microscope. We show that considering the global biomass of a cell population is a more robust and accurate method to assess its growth parameters in comparison to compiling individually segmented cells. This is especially true for confluent conditions. This method expands the use of light microscopy in answering biological questions concerning live cell populations even at high density. In contrast to labeling or lysis of cells this method does not alter the cells and could be useful in high-throughput screening and toxicity studies.
| S-EPMC7904828 | biostudies-literature
Project description:Continuous cell culture monitoring as a way of investigating growth, proliferation, and kinetics of biological experiments is in high demand. However, commercially available solutions are typically expensive and large in size. Digital inline-holographic microscopes (DIHM) can provide a cost-effective alternative to conventional microscopes, bridging the gap towards live-cell culture imaging. In this work, a DIHM is built from inexpensive components and applied to different cell cultures. The images are reconstructed by computational methods and the data are analyzed with particle detection and tracking methods. Counting of cells as well as movement tracking of living cells is demonstrated, showing the feasibility of using a field-portable DIHM for basic cell culture investigation and bringing about the potential to deeply understand cell motility.
| S-EPMC6427468 | biostudies-literature
Project description:BackgroundMulticellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.ResultsHere, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.ConclusionsAs illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.
| S-EPMC3274476 | biostudies-literature
Project description:BackgroundBecause of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures.MethodsWe demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump.Results & discussionImplemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.
| S-EPMC8239994 | biostudies-literature
Project description:Stimulated Raman scattering (SRS) microscopy is a powerful technique for visualising the cellular uptake and distribution of drugs and small molecules in live cells under biocompatible imaging conditions. The use of bio-orthogonal groups within the drug molecule, including alkynes and nitriles, has enabled the direct detection of a plethora of bioactive molecules in a minimally perturbative fashion. Limited progress has been made towards real-time detection of drug uptake and distribution into live cells under physiological conditions, despite the accordant potential it presents for preclinical drug development. SRS microscopy has been applied to the study of cellular dynamics of the drug 7RH, which is a potent inhibitor of dicoidin domain receptor 1 (DDR1) and prevents cellular adhesion, proliferation and migration in vitro. The uptake of 7RH into a variety of mammalian cell models was shown to be independent of DDR1 expression. Using a perfusion chamber, the recurrent treatment of live cancer cells was achieved, enabling 7RH uptake to be visualised in real-time using SRS microscopy, after which the viability of the same cellular population was assessed using commercially available fluorescent markers in a multimodal imaging experiment. The effect of 7RH treatment in combination with the chemotherapeutic, cisplatin was investigated using sequential perfusion and time-lapse imaging in the same live cell population, to demonstrate the application of the approach. SRS microscopy also identified potent inhibition of cellular adhesion and migration in breast cancer cell models with increasing 7RH treatment concentrations, thus representing a novel read-out methodology for phenotypic assays of this kind. The direct assessment of drug-cell interactions under physiological conditions offers significant potential for the preclinical drug development process.
| S-EPMC9428671 | biostudies-literature
Project description:Time-lapse or longitudinal fluorescence microscopy is broadly used in cell biology. However, current available time-lapse fluorescence microscopy systems are bulky and costly. The limited field-of-view (FOV) associated with the microscope objective necessitates mechanical scanning if a larger FOV is required. Here we demonstrate a wide FOV time-lapse fluorescence self-imaging Petri dish system, termed the Talbot Fluorescence ePetri, which addresses these issues. This system's imaging is accomplished through the use of the Fluorescence Talbot Microscopy (FTM). By incorporating a microfluidic perfusion subsystem onto the platform, we can image cell cultures directly from within an incubator. Our prototype has a resolution limit of 1.2 μm and an FOV of 13 mm(2). As demonstration, we obtained time-lapse images of HeLa cells expressing H2B-eGFP. We also employed the system to analyze the cells' dynamic response to an anticancer drug, camptothecin (CPT). This method can provide a compact and simple solution for automated fluorescence imaging of cell cultures in incubators.
| S-EPMC3587116 | biostudies-literature
Project description:Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24?hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1?kW cm(-2) at 640?nm for several minutes, the maximum dose at 405?nm is only ~50?J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.
| S-EPMC4611486 | biostudies-literature
Project description:Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.
| S-EPMC3197663 | biostudies-literature
Project description:In vitro quantification of the effect of mechanical loads on cells by live microscopy requires precise control of load and culture environment. Corresponding systems are often bulky, their setup and maintenance are time consuming, or the cell yield is low. Here, we show the design and initial testing of a new cell culture system that fits on standard light microscope stages. Based on the parallel plate principle, the system allows for live microscopy of cells exposed to flow-induced shear stress, features short setup time and requires little user interaction. An integrated feedback-controlled heater and a bubble trap enable long observation times. The key design feature is the possibility for quick exchange of the cultured cells. We present first test results that focus on verifying the robustness, biocompatibility, and ease of use of the device.
| S-EPMC6504681 | biostudies-other