Gaudenz Danuser

FRET or no FRET: a quantitative comparison.

Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.

Automatic fluorescent tag detection in 3D with super-resolution: application to the analysis of chromosome movement

In this paper, we describe an algorithmic framework for the automatic detection of diffraction‐limited fluorescent spots in 3D optical images at a separation below the Rayleigh limit, i.e. with super‐resolution. We demonstrate the potential of super‐resolution detection by tracking fluorescently tagged chromosomes during mitosis in budding yeast. Our biological objective is to identify and analyse the proteins responsible for the generation of tensile force during chromosome segregation. Dynamic measurements in living cells are made possible by green fluroescent protein (GFP)‐tagging chromosomes and spindle pole bodies to generate cells carrying four fluorescent spots, and observe the motion of the spots over time using 3D‐fluorescence microscopy. The central problem in spot detection arises with the partial or complete overlap of spots when tagged objects are separated by distances below the resolution of the optics. To detect multiple spots under these conditions, a set of candidate mixture models is built, and the best candidate is selected from the set based on χ2‐statistics of the residuals in least‐square fits of the models to the image data.

Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications.

Microcontact printing (microcP) is a cost effective and simple method to create chemically micropatterned surfaces for cell biological applications. We have combined the technique with the spontaneous molecular assembly of a polycationic PEG-grafted copolymer, poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG). PLL-g-PEG with omega-functionalized PEG chains was print-transferred onto tissue culture polystyrene (TCPS) or glass substrates, resulting in patterns with a lateral resolution down to 1 microm. Subsequently, dipping in an aqueous solution of non-functionalized PLL-g-PEG was used to backfill the non-printed regions of the surface, rendering them highly protein and thus cell resistant. In a second approach, proteins were stamped and a PLL-g-PEG backfill was applied for passivation of the bare surface regions. Printing of peptide(RGD)-functionalized PLL-g-PEG or proteins combined with a subsequent PLL-g-PEG backfill can be applied to a wide variety of substrate materials with negatively charged surfaces such as TCPS, glass and many metal oxides. We have tested the printed surfaces with human foreskin fibroblasts for cell adhesion and long-term performance and with fish epidermal keratocytes for cell motility and short-time behaviour. Both cell types reacted selectively to the surface micropatterns. Fibroblasts adhered to the printed (adhesive) regions only, where they remained attached up to at least 1 week and were even able to proliferate. Keratocyte spreading and motility were also directed by the geometry of the underlying patterns. The results prove that microcP in conjunction with the use of PLL-g-PEG and its derivatives provides a simple and robust alternative to previously reported micropatterning methods for future cell biological and biotechnological applications.

Recovery, Visualization, and Analysis of Actin and Tubulin Polymer Flow in Live Cells: A Fluorescent Speckle Microscopy Study

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.

THE STRAIN FIELD IN NORTHWESTERN GREECE AND THE IONIAN ISLANDS : RESULTS INFERRED FROM GPS MEASUREMENTS

Abstract Recent crustal movements detected by the analysis of repeated satellite geodetic measurements reflect the ongoing geodynamic processes in the Alpine-Mediterranean area. Superimposed on the large-scale counterclockwise rotation of the African plate, complex dynamic processes are affecting the lithospheric fragments between the African and Eurasian plates. Key features to better understand the driving forces and associated seismic activity in the Africa/Eurasia collision zone are the Calabrian and Hellenic arcs. In this paper geodynamic investigations along the West Hellenic arc are discussed. They are based on two epochs (1989 and 1993) of satellite geodetic measurements carried out using the US Global Positioning System (GPS). The results are presented in terms of relative displacements and strain rates. Within the time span of 4 years southwestern Greece has moved to the southwest relative to southeastern Italy by an average of 120 mm, increasing from 80 mm at Lefkada, in the center of the Ionian Islands, to 160 mm at the Peloponnesus. The maximum strain rate is 0.18 μstrain/a located in the vicinity of Lefkada, where anomalously high earthquake activity is observed. The data provide strong evidence for dextral strike-slip motion on the order of 25 mm/a along the Kephalonia Fault Zone (KFZ). The deformation field of the KFZ is interpreted as a transition zone between the kinematics of the Apulian platform and the West Hellenic fold and thrust belts.

Computational Analysis of F-Actin Turnover in Cortical Actin Meshworks Using Fluorescent Speckle Microscopy

Fluorescent speckle microscopy (FSM) is a new imaging technique with the potential for simultaneous visualization of translocation and dynamic turnover of polymer structures. However, the use of FSM has been limited by the lack of specialized software for analysis of the positional and photometric fluctuations of hundreds of thousand speckles in an FSM time-lapse series, and for translating this data into biologically relevant information. In this paper we present a first version of a software for automated analysis of FSM movies. We focus on mapping the assembly and disassembly kinetics of a polymer meshwork. As a model system we have employed cortical F-actin meshworks in live newt lung epithelial cells. We lay out the algorithm in detail and present results of our analysis. The high spatial and temporal resolution of our maps reveals a kinetic cycling of F-actin, where phases of polymerization alternate with depolymerization in a spatially coordinated fashion. The cycle rates change when treating cells with a low dose of the drug latrunculin A. This shows the potential of this technique for future quantitative screening of drugs affecting the actin cytoskeleton. Various control experiments demonstrate that the algorithm is robust with respect to intensity variations due to noise and photobleaching and that effects of focus plane drifts can be eliminated by manual refocusing during image acquisition.

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.

Automatic fluorescent tag localization II: improvement in super-resolution by relative tracking

We present an algorithm for the three‐dimensional (3D) tracking of multiple fluorescent subresolution tags with super‐resolution in images of living cells. Recently, we described an algorithm for the automatic detection of such tags in single frames and demonstrated its potential in a biological system. The algorithm presented here adds to the tag detector a module for relative tracking of the signals between frames. As with tag detection, the main problem in relative tracking arises when signals of multiple tags interfere. We propose a novel multitemplate matching framework that exploits knowledge of the microscope point spread function to separate the intensity contribution of each tag in image regions with signal interferences. We use this intensity splitting to reconstruct a template for each tag in the source frame and a patch in the target frame, which are both free of intensity contributions from other tag signals. Tag movements between frames are then tracked by seeking, for each template–patch pair, the displacement vector providing the best signal match in terms of the sum of squared intensity differences. Because template and patch generation of tags with overlapping signals are interdependent, the matching is carried out simultaneously for all tags, and in an iterative manner. We have examined the performance of our approach using synthetic 3D data and observed a significant increase in resolution and robustness as compared with our previously described detector. It is now possible to localize and track tags separated by a distance three times smaller than the Rayleigh limit with a relative positional accuracy of better than 50 nm. We have applied the new tracking system to extract metaphase trajectories of fluorescently tagged chromosomes relative to the spindle poles in budding yeast.

Quantitative fluorescent speckle microscopy: where it came from and where it is going

Fluorescent speckle microscopy (FSM) is a technology for analysing the dynamics of macromolecular assemblies. Originally, the effect of random speckle formation was discovered with microtubules. Since then, the method has been expanded to other proteins of the cytoskeleton such as f‐actin and microtubule binding proteins. Newly developed, specialized software for analysing speckle movement and photometric fluctuation in the context of polymer transport and turnover has turned FSM into a powerful method for the study of cytoskeletal dynamics in cell migration, division, morphogenesis and neuronal path finding. In all these settings, FSM serves as the quantitative readout to link molecular and genetic interventions to complete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering of molecular regulation of the cytoskeleton. Fully automated FSM assays can also be applied to live‐cell screens for toxins, chemicals, drugs and genes that affect cytoskeletal dynamics. We envision that FSM has the potential to become a core tool in automated, cell‐based molecular diagnostics in cases where variations in cytoskeletal dynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. In this paper, we review the origins of FSM, discuss these most recent technical developments and give a glimpse to future directions and potentials of FSM. It is written as a complement to the recent review (Waterman‐Storer & Danuser, 2002, Curr. Biol., 12, R633–R640), in which we emphasized the use of FSM in cell biological applications. Here, we focus on the technical aspects of making FSM a quantitative method.

New Directions for Fluorescent Speckle Microscopy

Fluorescent Speckle Microscopy (FSM) is a technology for analyzing cytoskeleton dynamics, giving novel insight into their roles in living cells. New applications of FSM, together with the development of computer-based FSM image analysis, will make FSM the first microscopy-based method to deliver quantitative kinetic readouts at high spatial and temporal resolution for a wide variety of macromolecular systems. Here, we review the most recent applications and developments and give a glimpse of future directions and potentials of FSM.