A novel sample holder for 4D live cell imaging to study cellular dynamics in complex 3D tissue cultures.
Ontology highlight
ABSTRACT:
SUBMITTER: Septiadi D
PROVIDER: S-EPMC6026119 | biostudies-literature | 2018 Jun
REPOSITORIES: biostudies-literature
Publications
A novel sample holder for 4D live cell imaging to study cellular dynamics in complex 3D tissue cultures.
Septiadi D D Bourquin J J Durantie E E Petri-Fink A A Rothen-Rutishauser B B
Scientific reports 20180629 1
Three dimensional (3D) co-cultures to mimic cellular dynamics have brought significant impacts in tissue engineering approaches for biomedical research. Herein, we present a novel sample holder combined with time-lapse fluorescence imaging technique, referred as 4D live cell imaging, allowing direct visualization of various cells up to 24 hours. We further extended our approach to monitor kinetics and dynamics of particle uptake by cells and translocation across tissue membranes. ...[more]
Similar Datasets
Project description:Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca(2+) and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms.
| S-EPMC4585674 | biostudies-literature
Project description:Epithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell-cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT-and the reverse mesenchymal-epithelial transition (MET)-are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell-cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos-gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos-that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer's vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET. Video Abstract.
| S-EPMC8296657 | biostudies-literature
Project description:A simple and cost effective alternative for fabricating custom Scanning Electron Microscope (SEM) sample holders using 3D printers and conductive polylactic acid filament is presented. The flexibility of the 3D printing process allowed for the fabrication of sample holders with specific features that enable the high-resolution imaging of nanoelectrodes and nanopipettes. The precise value of the inner semi cone angle of the nanopipettes taper was extracted from the acquired images and used for calculating their radius using electrochemical methods. Because of the low electrical resistivity presented by the 3D printed holder, the imaging of non-conductive nanomaterials, such as alumina powder, was found to be possible. The fabrication time for each sample holder was under 30 minutes and the average cost was less than $0.50 per piece. Despite being quick and economical to fabricate, the sample holders were found to be sufficiently resistant, allowing for multiple uses of the same holder.
| S-EPMC5533330 | biostudies-literature
Project description:Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon autofluorescence imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.
| S-EPMC7718391 | biostudies-literature
Project description:Tissue dynamics play critical roles in many physiological functions and provide important metrics for clinical diagnosis. Capturing real-time high-resolution 3D images of tissue dynamics, however, remains a challenge. This study presents a hybrid physics-informed neural network algorithm that infers 3D flow-induced tissue dynamics and other physical quantities from sparse 2D images. The algorithm combines a recurrent neural network model of soft tissue with a differentiable fluid solver, leveraging prior knowledge in solid mechanics to project the governing equation on a discrete eigen space. The algorithm uses a Long-short-term memory-based recurrent encoder-decoder connected with a fully connected neural network to capture the temporal dependence of flow-structure-interaction. The effectiveness and merit of the proposed algorithm is demonstrated on synthetic data from a canine vocal fold model and experimental data from excised pigeon syringes. The results showed that the algorithm accurately reconstructs 3D vocal dynamics, aerodynamics, and acoustics from sparse 2D vibration profiles.
| S-EPMC10199019 | biostudies-literature
Project description:It is estimated that two million new dental implants are inserted worldwide each year. Innovative implant materials are developed in order to minimize the risk of peri-implant inflammations. The broad range of material testing is conducted using standard 2D, terminal, and invasive methods. The methods that have been applied are not sufficient to monitor the whole implant surface and temporal progress. Therefore, we built a 3D peri-implant model using a cylindrical implant colonized by human gingival fibroblasts. In order to monitor the cell response over time, a non-toxic LIVE/DEAD staining was established and applied to the new 3D model. Our LIVE/DEAD staining method in combination with the time resolved 3D visualization using Scanning Laser Optical Tomography (SLOT), allowed us to monitor the cell death path along the implant in the 3D peri-implant model. The differentiation of living and dead gingival fibroblasts in response to toxicity was effectively supported by the LIVE/DEAD staining. Furthermore, it was possible to visualize the whole cell-colonized implant in 3D and up to 63 hours. This new methodology offers the opportunity to record the long-term cell response on external stress factors, along the dental implant and thus to evaluate the performance of novel materials/surfaces.
| S-EPMC6179276 | biostudies-literature
Project description:The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.
| S-EPMC3832664 | biostudies-literature
Project description:We report the first demonstration of a microfluidic platform that captures the full physiological range of mass transport in 3-D tissue culture. The basis of our method used long microfluidic channels connected to both sides of a central microtissue chamber at different downstream positions to control the mass transport distribution within the chamber. Precise control of the Péclet number (Pe), defined as the ratio of convective to diffusive transport, over nearly five orders of magnitude (0.0056 to 160) was achieved. The platform was used to systematically investigate the role of physiological mass transport on vasculogenesis. We demonstrate, for the first time, that vasculogenesis can be independently stimulated by interstitial flow (Pe > 10) or hypoxic conditions (Pe < 0.1), and not by the intermediate state (normal living tissue). This simple platform can be applied to physiological and biological studies of 3D living tissue followed by pathological disease studies, such as cancer research and drug screening.
| S-EPMC3510322 | biostudies-literature
Project description:The key to understanding, harnessing, and manipulating natural biological processes for the benefit of tissue engineering lies in providing a controllable dynamic environment for tissue development in vitro while being able to track cell activity in real time. This work presents a multi-channel bioreactor specifically designed to enable on-line imaging of fluorescently labeled cells embedded in replicated 3D engineered constructs subjected to different flow conditions. The images are acquired in 3D using a standard upright confocal microscope and further analyzed and quantified by computer vision. The platform is used to characterize and quantify the pace and directionality of angiogenic processes induced by flow. The presented apparatus bears considerable potential to advance scientific research, from basic research pursuing the effect of flow versus static conditions on 3D scaffolds and cell types, to clinically oriented modeling in drug screening and cytotoxicity assays.
| S-EPMC6499812 | biostudies-other
Project description:Bacteria have evolved numerous mechanisms for cell-cell communication, many of which have important consequences for human health. Among these is conjugation, the direct transfer of DNA from one cell to another. For gram-negative bacteria, conjugation requires thin, flexible filaments (conjugative pili) that are elaborated by DNA donor cells. The structure, function, and especially the dynamics of conjugative pili are poorly understood. Here, we have applied live-cell imaging to characterize the dynamics of F-pili (conjugative pili encoded by the F plasmid of Escherichia coli). We establish that F-pili normally undergo cycles of extension and retraction in the absence of any obvious triggering event, such as contact with a recipient cell. When made, such contacts are able to survive the shear forces felt by bacteria in liquid media. Our data emphasize the role of F-pilus flexibility both in efficiently sampling a large volume surrounding donor cells in liquid culture and in establishing and maintaining cell-cell contact. Additionally and unexpectedly, we infer that extension and retraction are accompanied by rotation about the long axis of the filament.
| S-EPMC2582581 | biostudies-literature