Optical flow analysis reveals that Kinesin-mediated advection impacts on the orientation of microtubules in the Drosophila oocyte.

The orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds; to counteract the recruitment of Kinesin to microtubules. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Light controlled motility of Escherichia coli. Characterization and applications

Characterization of wild type E. coli motility in response to light stimuli. Gene editing of bacteria to implement specifc functions (e.g. photokinesis). The engineered strain has been used to demonstrate that density modulation of photokinetic bacteria can be obtained by projecting spatially structured light on the sample. Additionally these bacteria have been also used as propelling units in microfabricated structures

Biomolecular Shuttles under Dielectrophoretic Forces

Motor proteins and filaments are essential elements in living cells. They are employed in skeletal muscles to generate forces, they transport cargos such as organelles to specific locations in the cells or they reorganize themselves to change a cell\u27s structure. Moreover, motor proteins and filaments use hydrolysis of adenosine triphosphate (ATP) as chemical fuel to generate mechanical movement in their interaction. Understanding the behavior of these enticed nano-sized machines and their properties, yet to be mimicked and synthesized by humans is very important to the future development of transport in nanoscale. Thus far, researchers succeeded in demonstrating the interaction of motor proteins and filaments in in vitro environment and controlling their random movement by various methods such as with the influence of DC electric field, driven flow field and engineered tracks by photolithographic method. In this thesis, dielectrophoretic forces, which are generated under nonuniform electric field by AC, are explored as a candidate to control the direction of biomolecular shuttles, actin filaments which glide on heavy meromyosin coated surface. Under dielectrophoretic forces, actin filaments showed bidirectional movement between embedded electrodes. The orientation and velocity of actin filaments were measured under various AC voltages, frequencies and distances between electrodes. Additionally, the effect of temperature on myosin-actin motility was further investigated and loading cargo on actin filaments was demonstrated by using a streptavidin-biotin binding system

Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

<p>Abstract</p> <p>Background</p> <p>Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions.</p> <p>Results</p> <p>We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells.</p> <p>Conclusion</p> <p>We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.</p

Automated measurement of cell motility and proliferation

BACKGROUND: Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. RESULTS: We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 um/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth. CONCLUSION: These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype

Laser and optical based methods for detecting and characterising microorganisms

This work investigated novel optical methods of characterizing the activity of microorganisms. Two different systems are studied in detail in this work. The possibility of using line scan speckle systems and imaging systems to understand the microbial behaviour, growth and motility was investigated. Conventionally, the growth and viability of microorganisms are determined by swabbing, plating and incubation, typically at 37degreesC for at least 24 hours. The proposed system allows real-time quantification of morphology and population changes of the microorganisms. An important aspect of the line scan system is the dynamic biospeckle. Dynamic speckle can be obtained from the movement of particles suspended in liquids. The speckle patterns show fluctuations in space and time which may be correlated with the activity of the constituents in the suspension. Initially the speckle parameters were standardized to non-motile and inert specimens such as polystyrene microspheres and suspensions of Staphylococcus aureus. The same optical systems and parameters were later tested on motile, active and live organisms of Escherichia coli. The experimental results that are presented describe the time history of the dynamic speckle pattern. A number of algorithms were used to analyse the intensity data. A 2D-FFT algorithm was used to evaluate the space and time-varying autocorrelation. Analysis of the speckle data in the Fourier domain provided insight into the motility of the organisms in broth. The mathematical analysis also gave further insight into the culture broth evaporation and its particle sedimentation characteristics at 37degreesC. These features correlated with the periodic motions associated with the organism and may therefore provide a signature for the organism and a means of monitoring. These results aided the developemnt of imaging bacterial detection systems which were discussed in the second half of the work. The second experimental system focuses on quantifying the morphology and population dynamics of Euglena gracilis under ambient conditions through image processing. Unlike many other cell systems, Euglena cells change from round to long to round cell shape and these different cell shapes were analyzed over time. In the morphological studies of single Euglena cells, image processing tools and filtering techniques were used and different parameters identified and their efficiency at determining cell shape compared. The best parameter for processing the images and its effectiveness in detecting even the interior motions of constituents within a dead cell was found. The efficiency of the measurement parameters in following sequences of shape changes of the Euglena cell was compared with the visual assessment tests from 12 volunteers and other simple measurement methods including parameters relating to the cells eccentricity, and image processing in the space and frequency domains. One of the major advantages of this system is that living cells can be examined in their natural state without being killed, fixed, and stained. As a result, the dynamics of ongoing biological processes in live cells can be observed and recorded in high contrast and sharp clarity. The population statistics of Euglena gracilis was done in liquid culture. A custom built microscopy system was employed and the laser beam was coupled with a dark field illumination system to enhance the contrast of the images. Different image filters were employed for extracting useful information on the population statistics. Similarly as with the shape study of the Euglena cell, different parameters were identified and the best parameter was selected. The population study of the Euglena cells provided a detection system that indicated the activity of the population

Multi-confocal Fluorescence Correlation Spectroscopy : experimental demonstration and potential applications for living cell measurements

We report, for the first time, a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique which allows parallel measurements at different locations, by combining a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM is calculated by using the spherical wave approximation and makes it possible to produce several diffraction limited laser spots, either aligned or spread over the field of view. To attain fast enough imaging rates, the camera has been used in different acquisition modes, the fastest of which leads to a time resolution of 100 $\mu$s. We qualified the experimental set-up by using solutions of sulforhodamine G in glycerol and demonstrated that the observation volumes are similar to that of a standard confocal set-up. To demonstrate that our mFCS method is suitable for intracellular studies, experiments have been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover, by analyzing the auto-correlation curves, the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to the complex network of interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock

Biophysics of DNA based Nanosystems Probed by Optical Nanoscopy

A dynamic DNA nanosystem exploits the programmable structure and energy landscape of DNA self-assembly to encode designed processes in a fuctuating molecular environment. One type of such a dynamic system, DNA walker, is reminiscent of biological motor proteins that convert chemical energy into mechanical translocation. Typical DNA walker travels tens of nanometers at a speed orders of magnitude slower than motor proteins. Two major challenges limited the development of functional DNA walkers. First, there are no suitable characterization methods that o˙er adequate spatial and temporal resolution to extract walker kinetics. Second, no guidelines have been established for the design and development of DNA walkers with specifed properties. In this work, an enzymatic DNA walker system that integrate oligonucleotides with nanomaterials is designed. This approach takes advantage of novel optical properties of nanomaterials and sub-di˙raction imaging techniques to study the kinetics and biophysical nature of synthetic DNA walkers. Design principles are extracted from walker kinetics for constructing functional walkers that can rival motor proteins. Multiple schemes are explored to regulate the walker motility so that various behaviors can be encoded into the system. This work demonstrates novel methods to design and construct molecular systems with programmed functions, which will pave the road for creating synthetic systems with encoded behaviors from the bottom up