Multi-field 3D scanning light microscopy of early embryogenesis.
ABSTRACT A computer-controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three-dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal- and positional-drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large-scale remodelling of the embryonic primordial vascular structure.
Article: A random cell motility gradient downstream of FGF controls elongation of an amniote embryo.[show abstract] [hide abstract]
ABSTRACT: Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient, which has been implicated in the control of cell motility in this tissue. Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data indicate that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements.Nature 07/2010; 466(7303):248-52. · 36.28 Impact Factor
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ABSTRACT: Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis.Cellular and Molecular Life Sciences CMLS 10/2010; 67(20):3489-97. · 6.57 Impact Factor
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ABSTRACT: The process required to track cellular structures is a key task in the study of cell migration. This allows the accurate estimation of motility indicators that help in the understanding of mechanisms behind various biological processes. This paper reports a particle-based fully automatic tracking framework that is able to quantify the motility of living cells in time-lapse images. Contrary to the standard tracking methods based on predefined motion models, in this paper we reformulate the tracking mechanism as a data driven optimization process to remove its reliance on a priory motion models. The proposed method has been evaluated using 2D and 3D deconvolved epifluorescent in-vivo image sequences that describe the development of the quail embryo. http://www.cipa.dcu.ie/pubs_full.htmlConference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 08/2011; 2011:6613-8.