On the Two-View Geometry of Unsynchronized Cameras

We present new methods for simultaneously estimating camera geometry and time shift from video sequences from multiple unsynchronized cameras. Algorithms for simultaneous computation of a fundamental matrix or a homography with unknown time shift between images are developed. Our methods use minimal correspondence sets (eight for fundamental matrix and four and a half for homography) and therefore are suitable for robust estimation using RANSAC. Furthermore, we present an iterative algorithm that extends the applicability on sequences which are significantly unsynchronized, finding the correct time shift up to several seconds. We evaluated the methods on synthetic and wide range of real world datasets and the results show a broad applicability to the problem of camera synchronization.Comment: 12 pages, 9 figures, Computer Vision and Pattern Recognition (CVPR) 201

Monitoring the Cytoskeletal EGF Response in Live Gastric Carcinoma Cells

<div><p>Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF) signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell’s motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.</p> </div

EGF induces ruffling in MKN1 responder cells.

<p>MKN1 and Hs746T cells were transfected to produce enhanced yellow fluorescence protein (YFP) under CMV-promoter control. The fluorescence was recorded by confocal laser scanning microscopy three times per minute for 10 min in the absence of EGF and 10 min after addition of EGF. (A, B) show the color-coded cell contour changes in the absence of EGF, (A’, B’) after addition of 5 ng/ml EGF. The data are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045280#pone.0045280.s001" target="_blank">Video S1</a>. Bar, 10 µm. The arrowheads indicate places where the wavelike movement of the cell border during ruffling can be discovered from the sequence of cell contours in MKN1 cells in the presence of EGF (compare A, A’). Note that such changes are not observed in Hs746T cells upon EGF treatment (B, B’). (C) Illustration of changes in cell contour for three time points to elucidate the measuring procedure of the periodic area change Δ_PA. The cell contour recorded at t = 0 is shown in black at top, at t = 1 in red (dashed line at top, solid line at bottom), and at t = 2 in green. The changes in cell area are highlighted in top by a striated area for the comparison of t = 0/t = 1 leading to |Δ₁| and below by checkered pattern for the comparison of t = 1/t = 2 (|Δ₂|). (D) Quantitative assessment of dynamic cell size changes in response to EGF. The histogram represents the periodic area change as measured from time-lapse series of fluorescence micrographs taken from MKN1 and Hs746T cells that had been labeled with YFP. Plotted is the relative increase in % of the measured value with respect to this value before addition of EGF (respectively for untreated and Cetuximab treated cells). Each set of measurements refers to a 10 min observation period with a recording frequency of 3 images/min. Cells were monitored in the absence of EGF (“untreated”; defined as 100%), in the presence of 5 ng/ml EGF (“EGF”), in the presence of 1 µg/ml Cetuximab (“Cetuximab”) and in the presence of both 5 ng/ml EGF and 1 µg/ml Cetuximab (“EGF + Cetuximab”). Note the increased EGF-dependent and Cetuximab-inhibitable ruffling activity (p-value determined by Student’s t-test) in MKN1 cells that is not discernible in Hs746T cells.</p

Zyxin partially co-distributes with dSH2 in focal adhesions and is recruited from zyxin-enriched fibrillar adhesion upon EGF treatment.

<p>MKN1 cells were co-transfected with constructs encoding RFP-zyxin and CFP-dSH2 (depicted in false green color). Note that both co-localize only partially in the more central parts of focal adhesions in the cell periphery (A, B). This is further highlighted by the line diagrams in A’ that were recorded along the arrow in A. In contrast, more centrally located focal adhesions and large fibrillar adhesions are predominantly positive for zyxin and contain very little dSH2 label (C, D). (D, D’) is taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045280#pone.0045280.s010" target="_blank">Video S10</a> and depicts a cell immediately before and 59 min after addition of 5 ng/ml EGF. Note the reduction of the very large zyxin-positive plaque-like areas (arrowheads in D) and the appearance of novel contacts in newly-formed lamella (arrowhead in D’). Bars, 2 µm in A, B; 5 µm in C, D.</p

Lamella differ from ruffles by the presence of focal adhesions.

<p>The images shown in A, A’ are taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045280#pone.0045280.s007" target="_blank">Video S7</a> and present overlays of DIC recordings and CFP-dSH2 fluorescence microscopy (false red color) in MKN1 cells. Note the induction of abundant, dSH2-negative ruffles (arrows) upon addition of 5 ng/ml EGF. The micrographs in B, B’ are taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045280#pone.0045280.s008" target="_blank">Video S8</a> that shows the overlays of YFP- (white) and paxillin-dsRed-fluorescence in a MKN1 cell before (B) and after addition of EGF (5 ng/ml). Formation and turnover of paxillin-positive focal adhesions are detectable in regions of newly-forming lamella (arrows). Bars, 10 µm.</p

Schematic summary of early EGF effects on cell motility and cytoskeletal remodeling.

<p>Note that the depicted cascade is initiated by EGF but that each of the successive steps is reversible and subject to additional regulation. (0) shows a single cell in the absence of EGF with mature focal adhesions, that are positive for paxillin, dSH2 and zyxin, in the cell periphery in black and fibrillar adhesions in the central part of the cell, that contain little dSH2 but are enriched in zyxin (red-framed black streaks). (1) After addition of EGF actin-rich juxtamembranous regions (red arrows) form highly dynamic ruffles and lamellipodia that are devoid of keratin filaments, microtubules and focal adhesions. (2) Membrane domains with high ruffling activity develop lamella. Lamella formation is linked to the appearance of focal complexes that contain paxillin but are negative for dSH2 and zyxin (gray). Microtubule plus-ends enter these newly-formed cell extensions (blue arrows and wavy line). (3) Focal complexes mature into focal adhesions by recruitment of zyxin and tyrosine phosphorylation. The keratin network subsequently extends into the lamella by integration of keratin filament precursors (green arrows) that nucleate in the vicinity of focal adhesions and are transported toward the cell interior in an actin-dependant fashion. After disassembly of fibrillar adhesions the polarized cell then translocates toward the leading edge (direction of migration indicated by white arrows) leaving behind retraction fibers at its rear (thin lines).</p

Triple labeling of MKN1 cells reveals sequential rearrangement of cytoskeletal filaments in response to EGF.

<p>MKN1 cells were co-transfected with expression constructs for mRFP-actin (A, A’, A”), EB3-CFP (B, B’, B”) and HK18-YFP (C, C’, C”) and were imaged before and after addition of 30 ng/ml EGF. Note that EGF-induced cell extensions are positive for actin and only later recruit microtubules (black arrows). These areas are still negative for keratin after 6 min of EGF treatment. Note also the co-alignment of actin bundles with microtubules (white arrows). A time-lapse sequence of the same cell beginning 10 min after EGF addition is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045280#pone.0045280.s005" target="_blank">Video S5</a>. Bar, 10 µm.</p