Biomedical subjects

G Danuser

Publications and source records attributed to G Danuser.

5 recordsLinked to original sources

PubMed2001

Super-resolution microscopy using normal flow decoding and geometric constraints.

Prior knowledge about the observed scene provides the key to restoration of frequencies beyond the bandpass of an imaging system (super-resolution). In conjunction with microscopy two super-resolution mechanisms have been mainly reported: analytic continuation of the frequency spectrum, and constrained image deconvolution. This paper describes an alternative approach to super-resolution. Prior knowledge is imposed through geometric and dynamic models of the scene. We illustrate our concept based on the stereo reconstruction of a micropipette moving in close proximity to a stationary target object. Information about the shape and the movement of the pipette is incorporated into the reconstruction algorithm. The algorithm was tested in a microrobot environment, where the pipette tip was tracked at sub-Rayleigh distances to the target. Based on the tracking results, a machine vision module controlled the manipulation of microscopic objects, e.g. latex beads or diamond mono-crystals. In the theoretical part of this paper we prove that knowledge of the form 'the pipette has moved between two consecutive frames of the movie' must result in a twofold increase in resolution. We used the normal flow of an image sequence to decode positional measures from motion evidence. In practice, super-resolution factors between 3 and 5 were obtained. The additional gain originates from the geometric constraints that were imposed upon the stereo reconstruction of the pipette axis.

Journal Article↗

PubMed2000

Probing f-actin flow by tracking shape fluctuations of radial bundles in lamellipodia of motile cells.

We examined the dynamics of radial actin bundles based on time-lapse movies of polarized light images of living neuronal growth cones. Using a highly sensitive computer vision algorithm for tracking, we analyzed the small shape fluctuations of radial actin bundles that otherwise remained stationary in their positions in the growth cone lamellipodium. Using the tracking software, we selected target points on radial bundles and measured both the local bundle orientations and the lateral displacements between consecutive movie frames. We found that the local orientation and the lateral displacement of a target point are correlated. The correlation can be explained using a simple geometric relationship between the lateral travel of tilted actin bundles and the retrograde flow of f-actin structures. Once this relationship has been established, we have turned the table and used the radial bundles as probes to measure the velocity field of f-actin flow. We have generated a detailed map of the complex retrograde flow pattern throughout the lamellipodium. Such two-dimensional flow maps will give new insights into the mechanisms responsible for f-actin-mediated cell motility and growth.

Actins↗

PubMed2000

Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones.

We investigated the motion of filopodia and actin bundles in lamellipodia of motile cells, using time-lapse sequences of polarized light images. We measured the velocity of retrograde flow of the actin network and the lateral motion of filopodia and actin bundles of the lamellipodium. Upon noting that laterally moving filopodia and actin bundles are always tilted with respect to the direction of retrograde flow, we propose a simple geometric model for the mechanism of lateral motion. The model establishes a relationship between the speed of lateral motion of actin bundles, their tilt angle with respect to the direction of retrograde flow, and the speed of retrograde flow in the lamellipodium. Our experimental results verify the quantitative predictions of the model. Furthermore, our observations support the hypothesis that lateral movement of filopodia is caused by retrograde flow of tilted actin bundles and by their growth through actin polymerization at the tip of the bundles inside the filopodia. Therefore we conclude that the lateral motion of tilted filopodia and actin bundles does not require a separate motile mechanism but is the result of retrograde flow and the assembly of actin filaments and bundles near the leading edge of the lamellipodium.

Actins↗

PubMed2000

Tracking differential interference contrast diffraction line images with nanometre sensitivity.

This paper presents a computer vision framework for detecting and tracking diffraction images of linear structures in differential interference contrast (DIC) microscopy. The tracker can resolve image displacements of 1/10 of a pixel despite the weak and orientation-dependent contrast in DIC, as well as the variable blur in such image data caused by vertical specimen movement. In our high numerical aperture, high magnification microscope set-up, this resolution corresponds to 5 nm in object space. In video DIC similar resolution has been reported hitherto only for rotationally symmetric targets such as bead images. The tracker was developed for measuring deflections of clamped microtubules with a freely moving second end. By analysing the thermal fluctuations of such microtubules it was possible to derive their elasticity. The paper describes a filtering scheme for the detection and localization of DIC diffraction line images which represent loci of microtubules. For tracking the movements of the extracted lines we adopted the sum of squared (brightness) differences algorithm from computer vision. The analysis of the fluctuation measurements demonstrates the high sensitivity of this tracking technique in quantifying positional and orientational changes. We derived that the theoretical limit in tracking displacements of such diffraction line images is 1.25 nm, four times below the experimentally verified sensitivity. This indicates that the proposed tracker is still suboptimal. Nevertheless, the tracking precision was sufficient to reveal subtle deviations in the distribution of microtubule deflection from free diffusion. They were induced by pivotal points and multiple positions of relaxation. Also, the results suggest that there were defects in the polymer structure which caused very small but significant bends in the microtubule axis.

Algorithms↗

PubMed1999

Pulsatile influxes of H+, K+ and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes.

Fluxes of H+, K+ and Ca2+ were measured with self-referencing ion-selective probes, near the plasma membrane of growing Lilium longiflorum pollen tubes. Measurements from three regions around short, steady-growing tubes showed small, steady influx of H+ over the distal 40 microm and a region of the tube within 50-100 microm of the grain with larger magnitude efflux from the grain. K+ fluxes were immeasurable in short tubes. Measurements of longer tubes that were growing in a pulsatile manner revealed a pulsatile influx of both H+ and K+ at the growing tip. The average fluxes at the cell surface during the peaks of the H+ and K+ pulses were 489+/-81 and 688+/-144 pmol cm-2 second-1, respectively. Growth was measured by tracking the pollen tips with a computer vision system that achieved a spatial resolution of approximately 1/10 pixel. The high spatial resolution enabled the detection of growth, and thus the changes in growth rates, with a temporal sampling rate of 1 frame/second. These data show that the H+ and K+ pulses have a phase lag of 103+/-9 and 100+/-11 degrees, respectively, with respect to the growth pulses. Calcium fluxes were also measured in growing tubes. During steady growth, the calcium influx was relatively steady. When pulsatile growth began, the basal Ca2+ influx decreased and a pulsatile component appeared, superimposed on the reduced basal Ca2+ flux. The peaks of the Ca2+ pulses at the cell surface averaged 38.4+/-2.5 pmol cm-2 second-1. Longer tubes had large pulsatile Ca2+ fluxes with smaller baseline fluxes. The Ca2+ influx pulses had a phase lag of 123+/-9 degrees with respect to the growth pulses.

Calcium↗