Quantitative measurement of cell migration using time-lapse videomicroscopy and non-linear system analysis (original) (raw)
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Bimodal Analysis of Mammary Epithelial Cell Migration in Two Dimensions
Annals of Biomedical Engineering, 2008
Cell migration paths of mammary epithelial cells (expressing different versions of the promigratory tyrosine kinase receptor Her2/Neu) were analyzed within a bimodal framework that is a generalization of the run-and-tumble description applicable to bacterial migration. The mammalian cell trajectories were segregated into two types of alternating modes, namely, the "directional-mode" (mode I, the more persistent mode, analogous to the bacterial run phase) and the "re-orientation-mode" (mode II, the less persistent mode, analogous to the bacterial tumble phase). Higher resolution (more pixel information, relative to cell size) and smaller sampling intervals (time between images) were found to give a better estimate of the deduced single cell dynamics (such as directional-mode time and turn angle distribution) of the various cell types from the bimodal analysis. The bimodal analysis tool permits the deduction of short-time dynamics of cell motion such as the turn angle distributions and turn frequencies during the course of cell migration compared to standard methods of cell migration analysis. We find that the twohour mammalian cell tracking data do not fall into the diffusive regime implying that the oftenused random motility expressions for mammalian cell motion (based on assuming diffusive motion) are invalid over the time steps (fraction of minute) typically used in modeling mammalian cell migration.
The motile behavior of human breast cancer cells characterized by time-lapse videomicroscopy
In Vitro Cellular & Developmental Biology - Animal, 1998
In the development of more effective therapeutic strategies for the prevention and treatment of various cancers, it is important to focus on biological characteristics that differentiate benign and malignant neoplasms. A key difference between benign and malignant tumors appears to be related to cellular motility and invasiveness. Since cancer cell movement is an important component of metastatic potential, various in vitro assays have been developed and used to study general cell movement, including chemoinvasion of coated membranes and phagokinetic tracks (1,8). To better define the relationship between cell motility and metastatic potential, the present study used time-lapse videomicroscopy and image analysis to compare and characterize the differences in motile behavior between two human breast cancer cell lines that have been used in numerous in vitro studies. These were MDA-MB-231 (MDA) cells, which display a high metastatic potential and MCF-7 cells, which have a low metastatic potential. Similarly, other investigators have used time-lapse videomicroscopy to quantify the motility of other cancer cells (4,6). MCF-7 human breast cancer cells were provided by Dr. Sam Brooks of the Michigan Cancer Foundation (Detroit, MI), MDA-MB-231 cells were obtained from ATCC (Rockville, MD) and the cells were grown as monolayer cultures in RPMI 1640 media as previously described (3). Cells were plated sparsely in T-25 flasks and their motility was recorded with phase-contrast optics at 400 X with a color video camera (Sony, Model CCD-IRIS/RGB). The microscope stage was maintained at a constant temperature (37 ° C) by a thermostat-controlled stage heater (Fryer, Model A-50-IN). The video signal was fed to a time-lapse videocassette recorder with a time compression ratio of 240:1 (Panasonic, Model AG-6730 S-VHS, Secaucus, N J). Phase-contrast images of each cell line were recorded for 9-10 h in 3-5 replicate experiments. In each recording the movement of 5-10 cells in a microscopic field was analyzed. At least 30 cells from each cell line were analyzed in this study. The video images, at different time intervals (10-60 rain) over a period of 4-8 h, were captured and digitized with video capture software (Apple Video Player, Videocapture) on a Macintosh computer (Quadra 630-AV). Computer-assisted image analysis was accomplished with the NIH Image 1.59 program. Cell contours of the digital images were manually outlined and their x,y coordinates, area, and perimeter were calculated with the NIH Image program. Parameters of cell motility and morphology that were calculated for each cell line included rate of movement, total path length, and perimeter index. Rate of cell movement was determined as the distance traveled by the centroid (geometric center) of individual cells divided by time (gm/h) and plotted at the midpoint between each time interval. Total path length was computed as the total path traveled by the cell centroid (in p.m) during the experimental period. Perimeter index (PI) was caiculated as the perimeter of the cell di-HEFERENCES
Automated Motion Analysis of Adherent Cells in Monolayer Culture
Lecture Notes in Computer Science, 2015
This paper presents a novel method for tracking and characterizing adherent cells in monolayer culture. A system of cell tracking employing computer vision techniques was applied to time-lapse videos of replicate normal human uro-epithelial cell cultures exposed to different concentrations of adenosine triphosphate (ATP), acquired over a 20 hour period. Subsequent analysis, comprising feature extraction, demonstrated the ability of the technique to successfully separate the modulated classes of cell.
An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration
Bio-protocol, 2021
In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte ® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuC...
Development, 2012
Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in threedimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and b-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes reformed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program.
Analytical Biochemistry - ANAL BIOCHEM, 1997
Single-cell assays of cell migration, while yielding dynamic measurements of cell position and morphology, are predominantly limited by the time required for data collection and analysis. Computer-aided fluorescence time-lapse videomicroscopy (CAFTiV) was developed in order to facilitate the tracking and rapid examination of large numbers of motile cells. The system combines time-lapse videomicroscopy with epifluorescence capability, which allows full automation of image capture, sorting, and analysis due to the low background in the fluorescence images. Utilizing the CAFTiV system, data analysis time was reduced from over 125 h to less than 1 labor minute. In addition, fluorescence imaging permits cell tracking in small-volume chambers (
Quantifying stretching and rearrangement in epithelial sheet migration
New Journal of Physics, 2013
Although understanding the collective migration of cells, such as that seen in epithelial sheets, is essential for understanding diseases such as metastatic cancer, this motion is not yet as well characterized as individual cell migration. Here we adapt quantitative metrics used to characterize the flow and deformation of soft matter to contrast different types of motion within a migrating sheet of cells. Using a finite-time Lyapunov exponent (FTLE) analysis, we find that-in spite of large fluctuations-the flow field of an epithelial cell sheet is not chaotic. Stretching of a sheet of cells (i.e. positive FTLE) is localized at the leading edge of migration and increases when the cells are more highly stimulated. By decomposing the motion of the cells into affine and non-affine components using the metric D 2 min , we quantify local plastic rearrangements and describe the motion of a group of cells in a novel way. We find an increase in plastic rearrangements with increasing cell densities, whereas inanimate systems tend to exhibit less non-affine rearrangements with increasing density.
Investigation of Cell Dynamics in Vitro by Time Lapse Microscopy and Image Analysis
Chemical engineering transactions, 2014
Pharmacological research is continuously working on the development of new drugs. This research typically starts from the formulation of new molecules that are first investigated at the cell scale, finally is completed with clinical trials. Investigation on the cell scale requires simple, reproducible and reliable assays, able to simulate physiological conditions in the lab. A wide range of biological processes, such as angiogenesis, inflammation, tissue regeneration, tumour growth and invasion, are strongly linked to cell proliferation and migration mechanisms that govern the dynamic evolution of both individual cells and cell aggregates. In this work we present an experimental methodology for the quantitative investigation of cell dynamics in vitro by live imaging of biological soft matter. Cell motility is observed by means of a Time Lapse Microscopy workstation, consisting of a motorized video-microscope equipped with an incubating system, and quantified by image analysis techniques. We report some preliminary experimental results relative to the migration of a tumour cell line both in random condition and in presence of an external stimulus, such as a chemical concentration gradient. The ultimate goal of this research is the development of a standard assay to be used as a test for drug efficiency, suitable for routine application in the pharmaceutical research.
Automated time-lapse microscopy and high-resolution tracking of cell migration
Cytotechnology, 2006
We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystal...
Migration of isogenic cell lines quantified by dynamic multivariate analysis of single-cell motility
Cell Adhesion & Migration, 2008
Cell migration is essential in many physiological and pathological processes. To understand this complex behavior, researchers have turned to quantitative, in vitro, image-based measurements to dissect the steps of cellular motility. With the rise of automated microscopy, the bottleneck in these approaches is no longer data acquisition, but data analysis. Using time-lapse microscopy and computer-assisted image analysis, we have developed a novel, quantitative assay that extracts a multivariate profile for cellular motility. This technique measures three dynamic parameters per single cell: speed, surface area, and an index of dynamic cell expansion/contraction activity (DECCA). Our assay can be used in combination with a variety of extracellular matrix components, or other soluble agents, to analyze the effects of the microenvironment on cellular migration dynamics in vitro. Our application was developed and tested using A431 and HT-1080 cell lines plated on laminin-332 or fibronectin substrates. Our results indicate that HT-1080 cells migrate faster, have a greater surface area, and have a higher DECCA index than A431 cells on both matrices (for all parameters, p < 0.05). Spearman's correlation coefficients suggest that for these cell lines and matrices, various combinations of the three measurements display low to medium-high levels of correlation. These findings compare well with previous literature. Our approach provides new tools to measure cellular migration dynamics and address questions on the relationship between cell motility and the microenvironment, using only common microscopy techniques, accessible image analysis applications, and a basic desktop computer for image processing.