Science.gov

Sample records for 3d time-lapse microscopy

  1. Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

    PubMed Central

    Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

    2016-01-01

    Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format. PMID:27886235

  2. Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

    NASA Astrophysics Data System (ADS)

    Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

    2016-11-01

    Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

  3. Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

    PubMed

    Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

    2016-11-25

    Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

  4. 3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics

    PubMed Central

    Sison, Miguel; Chakrabortty, Sabyasachi; Extermann, Jérôme; Nahas, Amir; James Marchand, Paul; Lopez, Antonio; Weil, Tanja; Lasser, Theo

    2017-01-01

    We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume. PMID:28230188

  5. 3D Time-lapse Imaging and Quantification of Mitochondrial Dynamics

    NASA Astrophysics Data System (ADS)

    Sison, Miguel; Chakrabortty, Sabyasachi; Extermann, Jérôme; Nahas, Amir; James Marchand, Paul; Lopez, Antonio; Weil, Tanja; Lasser, Theo

    2017-02-01

    We present a 3D time-lapse imaging method for monitoring mitochondrial dynamics in living HeLa cells based on photothermal optical coherence microscopy and using novel surface functionalization of gold nanoparticles. The biocompatible protein-based biopolymer coating contains multiple functional groups which impart better cellular uptake and mitochondria targeting efficiency. The high stability of the gold nanoparticles allows continuous imaging over an extended time up to 3000 seconds without significant cell damage. By combining temporal autocorrelation analysis with a classical diffusion model, we quantify mitochondrial dynamics and cast these results into 3D maps showing the heterogeneity of diffusion parameters across the whole cell volume.

  6. Chromatin Imaging with Time-Lapse Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    Time-lapse atomic force microscopy (AFM) is widely used for direct visualization of the nanoscale dynamics of various biological systems. The advent of high-speed AFM instrumentation made it possible to image the dynamics of proteins and protein-DNA complexes within millisecond time range. This chapter describes protocols for studies of structure and dynamics of nucleosomes with time-lapse AFM including the high-speed AFM instrument. The necessary specifics for the preparation of chromatin samples for imaging with AFM including the protocols for the surface preparation are provided. PMID:25827873

  7. Combined time-lapse cinematography and immuno-electron microscopy.

    PubMed

    Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

    1990-04-01

    A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

  8. 3D inversion of time-lapse CSEM data for reservoir monitoring

    NASA Astrophysics Data System (ADS)

    Black, N.; Wilson, G. A.; Zhdanov, M. S.

    2010-12-01

    Effective reservoir monitoring requires time-lapse reservoir information throughout the interwell volume. The ability to understand and control reservoir behavior over the course of production allows for optimization of reservoir performance and production strategies. Good monitoring information makes it possible to improve the timing and location of new drilling (for both production and injection wells), to recognize flow paths, and to map oil that has been bypassed. Recent studies have inferred the feasibility of time-lapse marine controlled-source electromagnetic (CSEM) methods for the monitoring of offshore oil and gas fields. However, quantitative interpretations to ascertain what reservoir information may be recovered have not been performed. The time-lapse CSEM inverse problem can be highly constrained since the geometry of the reservoir is established prior from high resolution seismic surveys, rock and fluid properties are measured from well logs, and multiple history matched production scenarios are contained in dynamic reservoir models. We present a 3D inversion study of synthetic time-lapse CSEM data modeled from dynamic reservoir simulations. We demonstrate that even with few constraints on the model, the hydrocarbon-water front can be recovered from 3D inversion.

  9. Probabilistic 3-D time-lapse inversion of magnetotelluric data: application to an enhanced geothermal system

    NASA Astrophysics Data System (ADS)

    Rosas-Carbajal, M.; Linde, N.; Peacock, J.; Zyserman, F. I.; Kalscheuer, T.; Thiel, S.

    2015-12-01

    Surface-based monitoring of mass transfer caused by injections and extractions in deep boreholes is crucial to maximize oil, gas and geothermal production. Inductive electromagnetic methods, such as magnetotellurics, are appealing for these applications due to their large penetration depths and sensitivity to changes in fluid conductivity and fracture connectivity. In this work, we propose a 3-D Markov chain Monte Carlo inversion of time-lapse magnetotelluric data to image mass transfer following a saline fluid injection. The inversion estimates the posterior probability density function of the resulting plume, and thereby quantifies model uncertainty. To decrease computation times, we base the parametrization on a reduced Legendre moment decomposition of the plume. A synthetic test shows that our methodology is effective when the electrical resistivity structure prior to the injection is well known. The centre of mass and spread of the plume are well retrieved. We then apply our inversion strategy to an injection experiment in an enhanced geothermal system at Paralana, South Australia, and compare it to a 3-D deterministic time-lapse inversion. The latter retrieves resistivity changes that are more shallow than the actual injection interval, whereas the probabilistic inversion retrieves plumes that are located at the correct depths and oriented in a preferential north-south direction. To explain the time-lapse data, the inversion requires unrealistically large resistivity changes with respect to the base model. We suggest that this is partly explained by unaccounted subsurface heterogeneities in the base model from which time-lapse changes are inferred.

  10. Analysis of the repeatability of time-lapse 3d vsp multicomponent surveys, delhi field

    NASA Astrophysics Data System (ADS)

    Carvalho, Mariana Fernandes de

    Delhi Field is a producing oil field located in northeastern Louisiana. In order to monitor the CO2 sweep efficiency, time-lapse 3D seismic data have been acquired in this area. Time-lapse studies are increasingly used to evaluate changes in the seismic response induced by the production of hydrocarbons or the injection of water, CO2 or steam into a reservoir. A 4D seismic signal is generated by a combination of production and injection effects within the reservoir as well as non-repeatability effects. In order to get reliable results from time-lapse seismic methods, it is important to distinguish the production and injection effects from the non-repeatability effects in the 4D seismic signal. Repeatability of 4D land seismic data is affected by several factors. The most significant of them are: source and receiver geometry inaccuracies, differences in seismic sources signatures, variations in the immediate near surface and ambient non-repeatable noise. In this project, two 3D multicomponent VSP surveys acquired in Delhi Field were used to quantify the relative contribution of each factor that can affect the repeatability in land seismic data. The factors analyzed in this study were: source and receiver geometry inaccura- cies, variations in the immediate near surface and ambient non-repeatable noise. This study showed that all these factors had a significant impact on the repeatability of the successive multicomponent VSP surveys in Delhi Field. This project also shows the advantages and disadvantages in the use of different repeata- bility metrics, normalized-root-mean-square (NRMS) difference and signal-to-distortion ratio (SDR) attribute, to evaluate the level of seismic repeatability between successive time-lapse seismic surveys. It is observed that NRMS difference is greatly influenced by time-shifts and that SDR attribute combined with the time-shift may give more distinct and representative repeatability information than the NRMS difference.

  11. Probabilistic 3-D time-lapse inversion of magnetotelluric data: Application to an enhanced geothermal system

    USGS Publications Warehouse

    Rosas-Carbajal, Marina; Linde, Nicolas; Peacock, Jared R.; Zyserman, F. I.; Kalscheuer, Thomas; Thiel, Stephan

    2015-01-01

    Surface-based monitoring of mass transfer caused by injections and extractions in deep boreholes is crucial to maximize oil, gas and geothermal production. Inductive electromagnetic methods, such as magnetotellurics, are appealing for these applications due to their large penetration depths and sensitivity to changes in fluid conductivity and fracture connectivity. In this work, we propose a 3-D Markov chain Monte Carlo inversion of time-lapse magnetotelluric data to image mass transfer following a saline fluid injection. The inversion estimates the posterior probability density function of the resulting plume, and thereby quantifies model uncertainty. To decrease computation times, we base the parametrization on a reduced Legendre moment decomposition of the plume. A synthetic test shows that our methodology is effective when the electrical resistivity structure prior to the injection is well known. The centre of mass and spread of the plume are well retrieved.We then apply our inversion strategy to an injection experiment in an enhanced geothermal system at Paralana, South Australia, and compare it to a 3-D deterministic time-lapse inversion. The latter retrieves resistivity changes that are more shallow than the actual injection interval, whereas the probabilistic inversion retrieves plumes that are located at the correct depths and oriented in a preferential north-south direction. To explain the time-lapse data, the inversion requires unrealistically large resistivity changes with respect to the base model. We suggest that this is partly explained by unaccounted subsurface heterogeneities in the base model from which time-lapse changes are inferred.

  12. Time-lapse 3D ground-penetrating radar during plot-scale infiltration experiments

    NASA Astrophysics Data System (ADS)

    Allroggen, Niklas; Jackisch, Conrad; Tronicke, Jens

    2016-04-01

    In electrical resistive soils, surface-based ground-penetrating radar (GPR) is known as the geophysical tool providing the highest spatial resolution. Thus, 2D and 3D GPR surveys are commonly used for imaging subsurface structures or estimating soil moisture content. Due to its sensitivity to soil moisture and its non-invasive character, GPR provides a large potential to monitor soil moisture variation at high temporal and spatial resolution. As shown in previous experiments, the acquisition of time-lapse GPR data under field conditions requires a high data quality in terms of repeatability as well as spatial and temporal resolution. We present hydrogeophysical field experiments at the plot scale (1m x 1m), during which we record time-lapse 3D GPR. For GPR data acquisition, we use a pulseEKKO PRO GPR system equipped with a pair of 500 MHz antennas in combination with a specially designed metal-free measuring platform. Additionally, we collect tracer and soil moisture data, which are used to improve the interpretation of the GPR data with special focus on preferential flow paths and their structured advective flow field. After an accurate time-lapse GPR data processing, we compare 3D reflection events before and after infiltration and quantitatively interpret their relative time-shift in terms of soil moisture variations. Thereby, we are able to account for basically all of the infiltrated water. The first experiments demonstrate the general applicability of our experimental approach but are limited by the number of acquired time steps and measurement during the sprinkling period (the time of the highest temporal dynamics) are not possible at all. Based on this experience we redesign our experimental setup to continuously collect GPR data during irrigation and infiltration. Thereby, we strongly increase the temporal resolution of our measurements, improve the interpretability of the GPR data, and monitor the temporal and spatial dynamics of shallow subsurface

  13. Time-lapse microscopy using smartphone with augmented reality markers.

    PubMed

    Baek, Dongyoub; Cho, Sungmin; Yun, Kyungwon; Youn, Keehong; Bang, Hyunwoo

    2014-04-01

    A prototype system that replaces the conventional time-lapse imaging in microscopic inspection for use with smartphones is presented. Existing time-lapse imaging requires a video data feed between a microscope and a computer that varies depending on the type of image grabber. Even with proper hardware setups, a series of tedious and repetitive tasks is still required to relocate to the region-of-interest (ROI) of the specimens. In order to simplify the system and improve the efficiency of time-lapse imaging tasks, a smartphone-based platform utilizing microscopic augmented reality (μ-AR) markers is proposed. To evaluate the feasibility and efficiency of the proposed system, a user test was designed and performed, measuring the elapse time for a trial of the task starting from the execution of the application software to the completion of restoring and imaging of an ROI saved in advance. The results of the user test showed that the average elapse time was 65.3 ± 15.2 s with 6.86 ± 3.61 μm of position error and 0.08 ± 0.40 degrees of angle error. This indicates that the time-lapse imaging task was accomplished rapidly with a high level of accuracy. Thus, simplification of both the system and the task was achieved via our proposed system.

  14. Time-lapse 3-D seismic imaging of shallow subsurface contaminant flow.

    PubMed

    McKenna, J; Sherlock, D; Evans, B

    2001-12-01

    This paper presents a physical modelling study outlining a technique whereby buoyant contaminant flow within water-saturated unconsolidated sand was remotely monitored utilizing the time-lapse 3-D (TL3-D) seismic response. The controlled temperature and pressure conditions, along with the high level of acquisition repeatability attainable using sandbox physical models, allow the TL3-D seismic response to pore fluid movement to be distinguished from all other effects. TL3-D seismic techniques are currently being developed to monitor hydrocarbon reserves within producing reservoirs in an endeavour to improve overall recovery. However, in many ways, sandbox models under atmospheric conditions more accurately simulate the shallow subsurface than petroleum reservoirs. For this reason, perhaps the greatest application for analogue sandbox modelling is to improve our understanding of shallow groundwater and environmental flow mechanisms. Two fluid flow simulations were conducted whereby air and kerosene were injected into separate water-saturated unconsolidated sand models. In both experiments, a base 3-D seismic volume was recorded and compared with six later monitor surveys recorded while the injection program was conducted. Normal incidence amplitude and P-wave velocity information were extracted from the TL3-D seismic data to provide visualization of contaminant migration. Reflection amplitudes displayed qualitative areal distribution of fluids when a suitable impedance contrast existed between pore fluids. TL3-D seismic reflection tomography can potentially monitor the change in areal distribution of fluid contaminants over time, indicating flow patterns. However, other research and this current work have not established a quantifiable relationship between either normal reflection amplitudes and attenuation and fluid saturation. Generally, different pore fluids will have unique seismic velocities due to differences in compressibility and density. The predictable

  15. Time-lapse 3D VSP monitoring of a carbon dioxide injection project at Delhi Field, Louisiana

    NASA Astrophysics Data System (ADS)

    Lubis, Muhammad Husni Mubarak

    Delhi Field is a producing oil field located in northeastern Louisiana. The estimated original oil in place (OOIP) is 357 mmbo and approximately 54% of OOIP has been produced through the primary production and water-flooding. A CO2-EOR program has been implemented since November 2009 to recover an additional 17% of OOIP. Reservoir surveillance using time-lapse 3D seismic data has been conducted to monitor the CO2 sweep efficiency. The goal of this study is to monitor the CO2 flow-path in the area around the injector using time-lapse 3D VSP data. For this purpose, two 3D VSPs acquired in June 2010 and again in August 2011 were processed together. Fluid substitution and VSP modeling were performed to understand the influence of pore-fluid saturation change on VSP records. A cross-equalization was performed to improve the similarity of the datasets. This step is important to reduce the ambiguity in time-lapse observation. The splice of a 3D VSP image into the surface seismic data becomes the key point in determining the reflector of the reservoir. By integrating the observation from the modeling and the splice of 3D VSP image to surface seismic, the CO2 flow-path from injector 164-3 can be identified from 3D time-lapse VSP data. The CO2 was not radially distributed around the injector, but moved toward southwest direction. This finding is also consistent with the flow-path interpreted from surface seismic. This consistency implies that time-lapse 3D VSP surveys at Delhi Field confirm and augment the time-lapse interpretation from surface seismic data.

  16. A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics

    PubMed Central

    Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

    2016-01-01

    Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community. PMID:28002463

  17. A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics.

    PubMed

    Hernández Vera, Rodrigo; Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

    2016-01-01

    Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

  18. Time lapse 3D geoelectric measurements for monitoring of in-situ remediation

    NASA Astrophysics Data System (ADS)

    Tildy, Péter; Neducza, Boriszláv; Nagy, Péter; Kanli, Ali Ismet; Hegymegi, Csaba

    2017-01-01

    In the last decade, different kinds of in-situ methods have been increasingly used for hydrocarbon contamination remediation due to their effectiveness. One of these techniques operates by injection of chemical oxidant solution to remove (degrade) the subsurface contaminants. Our aim was to develop a surface (non-destructive) measurement strategy to monitor oxidative in-situ remediation processes. The difficulties of the presented study originate from the small volume of conductive solution that can be used due to environmental considerations, the effect of conductive groundwater and the high clay content of the targeted layer. Therefore a site specific synthetic modelling was necessary for measurement design involving the results of preliminary 2D ERT measurements, electrical conductivity measurements of different active agents and expected resistivity changes calculated by soil resistivity modelling. The results of soil resistivity modelling have suggested that the reagent have complex effects on contaminated soils because of chemical biodegradation. As a result the plume of resistivity changes caused by the injected agent was determined showing strong fracturing effect because of the high pressure of injection. Based on the sophisticated tests and synthetic modelling 3D time-lapse geo-electric measurements were proven to provide a usable monitoring tool for in-situ remediation to help in-field design of such techniques.

  19. Time-lapse 3D electrical resistivity tomography to monitor soil-plant interactions

    NASA Astrophysics Data System (ADS)

    Boaga, Jacopo; Rossi, Matteo; Cassiani, Giorgio; Putti, Mario

    2013-04-01

    In this work we present the application of time-lapse non-invasive 3D micro- electrical tomography (ERT) to monitor soil-plant interactions in the root zone in the framework of the FP7 Project CLIMB (Climate Induced Changes on the Hydrology of Mediterranean Basins). The goal of the study is to gain a better understanding of the soil-vegetation interactions by the use of non-invasive techniques. We designed, built and installed a 3D electrical tomography apparatus for the monitoring of the root zone of a single apple tree in an orchard located in the Trentino region, Northern Italy. The micro-ERT apparatus consists of 48 buried electrodes on 4 instrumented micro boreholes plus 24 mini-electrodes on the surface spaced 0.1 m on a square grid. We collected repeated ERT and TDR soil moisture measurements for one year and performed two different controlled irrigation tests: one during a very dry Summer and one during a very wet and highly dynamic plant growing Spring period. We also ran laboratory analyses on soil specimens, in order to evaluate the electrical response at different saturation steps. The results demonstrate that 3D micro-ERT is capable of characterizing subsoil conditions and monitoring root zone activities, especially in terms of root zone suction regions. In particular, we note that in very dry conditions, 3D micro ERT can image water plumes in the shallow subsoil produced by a drip irrigation system. In the very dynamic growing season, under abundant irrigation, micro 3D ERT can detect the main suction zones caused by the tree root activity. Even though the quantitative use of this technique for moisture content balance suffers from well-known inversion difficulties, even the pure imaging of the active root zone is a valuable contribution. However the integration of the measurements in a fully coupled hydrogeophysical inversion is the way forward for a better understanding of subsoil interactions between biomass, hydrosphere and atmosphere.

  20. Contribution of 3-D time-lapse ERT to the study of leachate recirculation in a landfill

    SciTech Connect

    Clement, R.; Oxarango, L.; Descloitres, M.

    2011-03-15

    Leachate recirculation is a key process in the operation of municipal waste landfills as bioreactors. It aims at increasing the moisture content to optimise the biodegradation. Because waste is a very heterogeneous and anisotropic porous media, the geometry of the leachate plume recirculation is difficult to delineate from the surface at the scale of the bioreactor site. In this study, 3-D time-lapse electrical resistivity tomography (ERT) was used to obtain useful information for understanding leachate recirculation hydrodynamics. The ERT inversion methodology and the electrode arrays were optimised using numerical modelling simulating a 3-D leachate injection scenario. Time-lapse ERT was subsequently applied at the field scale during an experimental injection. We compared ERT images with injected volumes to evaluate the sensitivity of time-lapse ERT to delineate the plume migration. The results show that time-lapse ERT can accomplish the following: (i) accurately locate the injection plume, delineating its depth and lateral extension; (ii) be used to estimate some hydraulic properties of waste.

  1. Time-lapse analysis of sparse 3D seismic data from the CO2 storage pilot site at Ketzin, Germany

    NASA Astrophysics Data System (ADS)

    Ivandic, M.; Yang, C.; Lüth, S.; Cosma, C.; Juhlin, C.

    2012-04-01

    Capture and geological storage of CO2 is considered to be a feasible method for reducing carbon emissions. In April 2004, a research pilot project in the German town of Ketzin started as the first onshore CO2 storage project in Europe. Injection started in June 2008 and until the latest repeat survey in February 2011 around 45 kilotons of CO2 had been injected into a saline aquifer at approximately 630-650 m depth. Different seismic methods, such as time-lapse Vertical Seismic Profiling (VSP), Crosswell, Moving Source Profiling (MSP) and surface seismics have been employed to detect and monitor changes in the reservoir. We present here time-lapse results from sparse 3D seismic surveying with a "star" geometry, i.e. with a radial distribution of acquisition profiles directed towards the approximate location of the injection well, which were acquired to link downhole surveys with full 3D surface seismic surveys. The main objectives of the sparse 3D surveys were (1) to identify changes in the seismic response related to the injection of CO2 between the repeat surveys and baseline survey and (2) to compare these results with those from the repeat 3D seismic survey. The results are consistent with the 3D seismic time-lapse studies over the injection site and show that the sparse 3D geometry can be used to qualitatively map the migration of the CO2 plume within the saline reservoir, as well as potential migration out of the reservoir rock at a significantly lower effort than the full 3D surveying. The latest repeat survey indicates preferential migration of the CO2 to the west. Both sparse 3D repeat surveys show that the CO2 is being confined within the aquifer, implying that there is no leakage into the caprock at the time of the repeat surveys. The same observation was obtained from the 3D dataset.

  2. Enhanced imaging of CO2 at the Ketzin storage site: Inversion of 3D time-lapse seismic data

    NASA Astrophysics Data System (ADS)

    Gil, M.; Götz, J.; Ivanova, A.; Juhlin, C.; Krawczyk, C. M.; Lüth, S.; Yang, C.

    2012-04-01

    The Ketzin test site, located near Berlin, is Europe's longest-operating on-shore CO2 storage site. As of December 2011, more than 56,000 tons of food grade CO2 has been injected since June 2008 in an anticlinal structure of the Northeast German Basin. The target reservoir consists of porous, brine bearing sandstone units of the Upper Triassic Stuttgart Formation at approximately 630 to 650 m depth. In order to enhance the understanding of the structural geometry of the site and to investigate the extension of the CO2-plume, several geophysical monitoring methods are being applied at Ketzin, among these are active seismic measurements, geoelectrics and borehole measurements. Among the various seismic techniques (e.g. 2D reflection surveys, crosshole tomography, Vertical Seismic Profiling, 2D- and 3D-Moving Source Profiling) employed at this pilot site, 3D time-lapse reflection surveys are an important component. The baseline 3D survey was acquired in 2005 and the first repeat measurements were performed in 2009 after injection of about 22,000 tons of CO2. The second repeat survey is planned to be carried out in fall 2012. These measurements allow the time-lapse signature of the injected CO2 to be imaged. The time-lapse amplitude variation attributed to the injected CO2 in the reservoir matches, considering detection limits of seismic surface measurements, the expected distribution of the CO2 plume derived from reservoir simulations. Previous attempts towards a quantitative interpretation were based on integrative considerations of different types of geophysical measurements using strict assumptions and characterized by large error bars. In order to increase the resolution and reliability of the data and to improve estimation of rock properties and especially to enhance the imaging resolution of the CO2-plume, the time-lapse 3D seismic data have now been inverted for seismic impedances with different methods, which is the focus of this presentation. One difficulty

  3. Automated time-lapse microscopy and high-resolution tracking of cell migration

    SciTech Connect

    Fotos, Joseph S.; Vivek, Patel P.; Karin, Norm J.; Temburni, Murali; Koh, John T.; Galileo, Deni S.

    2006-08-09

    The study of cell motility is greatly enhanced by using a fully-automated high-throughput time-lapse microscopy system that is capable of collecting and analyzing data (1) from closely-spaced time points (seconds to minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) under several different experimental conditions simultaneously. Time-lapse video images collected under phase contrast and fluorescent illumination were analyzed using parameters of migration velocity, total accumulated distance (path length), and directionality for individual cells or for averaged cell populations. Quantitation of ''scratch'' or ''wound healing'' assays revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells with high resolution and, thus, is a vast distinct improvement of current methods. Several fluorescent vital labeling methods commonly used for end-point analyses, including GFP expression, were evaluated and most were useful for time-lapse studies under specific conditions. For example, fluorescently-labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules displayed altered migration velocities compared to tumor cells plated directly onto culture dishes. The techniques described here revealed cell motility behavior not discernable by previously-used methods. We propose that quantitative time-lapse video analysis will foster the creation new cell motility assays, and increase the resolution and accuracy of existing assays.

  4. Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

    PubMed Central

    Mazo-Vargas, Anyimilehidi; Park, Heungwon; Aydin, Mert; Buchler, Nicolas E.

    2014-01-01

    Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression. PMID:25232010

  5. Long-term time-lapse multimodal microscopy for tracking cell dynamics in live tissue

    NASA Astrophysics Data System (ADS)

    Graf, Benedikt W.; Valero, Maria C.; Chaney, Eric J.; Marjanovic, Marina; Boppart, Marni D.; Boppart, Stephen A.

    2011-02-01

    High speed intravital microscopy has emerged as an essential tool for studying cellular dynamics in live tissue. A limitation of this technique, however, is that the timescale that a sample can be continuously imaged is limited by practical considerations to several hours. Long term observation of live tissue is of great interest for a variety of research areas. We present methods for observing long term cellular dynamics in live tissue based on three-dimensional registration of time-lapse intravital microscopy images. For these experiments we utilized a custom multimodal microscope that allows simultaneous and co-registered acquisition of optical coherence (OCM) and multiphoton (MPM) microscopy images. OCM allows the structure of a sample to be visualized based on backscattered light while MPM excited fluorescence allows individual cells and cell function to be visualized. The OCM images of tissue structure are used to register data sets taken at different time points. The transformations of the OCM images are applied to MPM images to determine the migration of cell populations. This method of image registration is applied to in vivo tracking of bone-marrow derived GFP-labeled stem cells in mouse skin following bone marrow transplants from GFP donors into species-matched wildtype hosts. The use of three-dimensional image registration of time-lapse microscopy images enables tracking these cells after local cutaneous injury, and for investigating the role of skin stem cells in wound healing.

  6. Time-lapse microscopy patent upheld in Europe: response to Pearce.

    PubMed

    Sterckx, Sigrid; Cockbain, Julian; Pennings, Guido

    2017-02-01

    In this piece, we comment on the article by Pearce earlier in this journal. As Pearce correctly points out, what is fundamentally at issue in ESHRE et al's opposition to Stanford University's European patent on time-lapse microscopy is whether an exclusion from patentability, here of methods of medical diagnosis, should be interpreted narrowly or not. In the present case, the dominant piece of case law from the European Patent Office (EPO) gives a narrow interpretation of what a method of diagnosis must be in order not to be patentable. In their submissions to the EPO, ESHRE et al. have argued that this narrow interpretation is unfounded and incorrect.

  7. Estimation and Discrimination of Stochastic Biochemical Circuits from Time-Lapse Microscopy Data

    PubMed Central

    Thorsley, David; Klavins, Eric

    2012-01-01

    The ability of systems and synthetic biologists to observe the dynamics of cellular behavior is hampered by the limitations of the sensors, such as fluorescent proteins, available for use in time-lapse microscopy. In this paper, we propose a generalized solution to the problem of estimating the state of a stochastic chemical reaction network from limited sensor information generated by microscopy. We mathematically derive an observer structure for cells growing under time-lapse microscopy and incorporates the effects of cell division in order to estimate the dynamically-changing state of each cell in the colony. Furthermore, the observer can be used to discrimate between models by treating model indices as states whose values do not change with time. We derive necessary and sufficient conditions that specify when stochastic chemical reaction network models, interpreted as continuous-time Markov chains, can be distinguished from each other under both continual and periodic observation. We validate the performance of the observer on the Thattai-van Oudenaarden model of transcription and translation. The observer structure is most effective when the system model is well-parameterized, suggesting potential applications in synthetic biology where standardized biological parts are available. However, further research is necessary to develop computationally tractable approximations to the exact generalized solution presented here. PMID:23139740

  8. Time Lapse of World’s Largest 3-D Printed Object

    SciTech Connect

    2016-08-29

    Researchers at the MDF have 3D-printed a large-scale trim tool for a Boeing 777X, the world’s largest twin-engine jet airliner. The additively manufactured tool was printed on the Big Area Additive Manufacturing, or BAAM machine over a 30-hour period. The team used a thermoplastic pellet comprised of 80% ABS plastic and 20% carbon fiber from local material supplier. The tool has proven to decrease time, labor, cost and errors associated with traditional manufacturing techniques and increased energy savings in preliminary testing and will undergo further, long term testing.

  9. Infrared Time Lapse of World’s Largest 3D-Printed Object

    SciTech Connect

    2016-08-29

    Researchers at Oak Ridge National Laboratory have 3D-printed a large-scale trim tool for a Boeing 777X, the world’s largest twin-engine jet airliner. The additively manufactured tool was printed on the Big Area Additive Manufacturing, or BAAM machine over a 30-hour period. The team used a thermoplastic pellet comprised of 80% ABS plastic and 20% carbon fiber from local material supplier. The tool has proven to decrease time, labor, cost and errors associated with traditional manufacturing techniques and increased energy savings in preliminary testing and will undergo further, long term testing.

  10. CO2 mass estimation visible in time-lapse 3D seismic data from a saline aquifer and uncertainties

    NASA Astrophysics Data System (ADS)

    Ivanova, A.; Lueth, S.; Bergmann, P.; Ivandic, M.

    2014-12-01

    At Ketzin (Germany) the first European onshore pilot scale project for geological storage of CO2 was initiated in 2004. This project is multidisciplinary and includes 3D time-lapse seismic monitoring. A 3D pre-injection seismic survey was acquired in 2005. Then CO2 injection into a sandstone saline aquifer started at a depth of 650 m in 2008. A 1st 3D seismic repeat survey was acquired in 2009 after 22 kilotons had been injected. The imaged CO2 signature was concentrated around the injection well (200-300 m). A 2nd 3D seismic repeat survey was acquired in 2012 after 61 kilotons had been injected. The imaged CO2 signature further extended (100-200 m). The injection was terminated in 2013. Totally 67 kilotons of CO2 were injected. Time-lapse seismic processing, petrophysical data and geophysical logging on CO2 saturation have allowed for an estimate of the amount of CO2 visible in the seismic data. This estimate is dependent upon a choice of a number of parameters and contains a number of uncertainties. The main uncertainties are following. The constant reservoir porosity and CO2 density used for the estimation are probably an over-simplification since the reservoir is quite heterogeneous. May be velocity dispersion is present in the Ketzin reservoir rocks, but we do not consider it to be large enough that it could affect the mass of CO2 in our estimation. There are only a small number of direct petrophysical observations, providing a weak statistical basis for the determination of seismic velocities based on CO2 saturation and we have assumed that the petrophysical experiments were carried out on samples that are representative for the average properties of the whole reservoir. Finally, the most of the time delay values in the both 3D seismic repeat surveys within the amplitude anomaly are near the noise level of 1-2 ms, however a change of 1 ms in the time delay affects significantly the mass estimate, thus the choice of the time-delay cutoff is crucial. In spite

  11. In situ visualization of magma deformation at high temperature using time-lapse 3D tomography

    NASA Astrophysics Data System (ADS)

    Godinho, jose; Lee, Peter; Lavallee, Yan; Kendrick, Jackie; Von-Aulock, Felix

    2016-04-01

    We use synchrotron based x-ray computed micro-tomography (sCT) to visualize, in situ, the microstructural evolution of magma samples 3 mm diameter with a resolution of 3 μm during heating and uniaxial compression at temperatures up to 1040 °C. The interaction between crystals, melt and gas bubbles is analysed in 4D (3D + time) during sample deformation. The ability to observe the changes of the microstructure as a function of time allow us to: a) study the effect of temperature in the ability of magma to fracture or deform; b) quantify bubble nucleation and growth rates during heating; c) study the relation between crystal displacement and volatile exsolution. We will show unique beautiful videos of how bubbles grow and coalescence, how samples and crystals within the sample fracture, heal and deform. Our study establishes in situ sCT as a powerful tool to quantify and visualize with micro-scale resolution fast processes taking place in magma that are essential to understand ascent in a volcanic conduit and validate existing models for determining the explosivity of volcanic eruptions. Tracking simultaneously the time and spatial changes of magma microstructures is shown to be primordial to study disequilibrium processes between crystals, melt and gas phases.

  12. Evaluation and localization of an artificial drainage network by 3D time-lapse electrical resistivity tomography.

    PubMed

    Jouen, T; Clément, R; Henine, H; Chaumont, C; Vincent, B; Tournebize, J

    2016-08-26

    In France, 10 % of total arable land is equipped with subsurface drainage systems, to control winter and spring waterlogging due to a temporary perched water table. Most of these systems were installed in the1980s and have aged since then and may now need maintenance. Sometimes, the location of the systems is known, but the standard situation in France is that the original as-built master sketches are no longer available. Performance assessment of drainage systems and curative actions are complicated since drain location is unknown. In this article, the authors test the application of a non-destructive drain detection method which consists in water injection at the outfall of the drainage network combined with time-lapse electrical resistivity tomography (ERT) monitoring. To assess the performance of this methodology, which consists in measuring electrical resistivity from electrodes placed at the nodes of a 1.2-m regular mesh, the authors interpreted the signal using a two-step approach. The first step is based on 3D ERT numerical modelling during a scenario of surface infiltration processes (forward modelling followed by geophysical inversion); this step optimizes the ERT method for locating the infiltration at depths below 1 m. The second step is the validation of the results obtained by numerical modelling with an experimental data set, using water injection into the drainage network combined with time-lapse ERT monitoring on an experimental field site. The results showed the relevance of time-lapse ERT monitoring on a small agricultural plot for locating the drainage network. The numerical results also showed several limitations of the combined methodology: (i) it is necessary to use an electrode spacing unit less than 1.20 m, which does not facilitate investigation on large agriculture plots, (ii) measurements must be taken when resistivity contrast is the strongest between the infiltration area and the soil and (iii) the volume of water needed for

  13. Watching amyloid fibrils grow by time-lapse atomic force microscopy.

    PubMed

    Goldsbury, C; Kistler, J; Aebi, U; Arvinte, T; Cooper, G J

    1999-01-08

    Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

  14. Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

    PubMed Central

    de Jong, Imke G.; Beilharz, Katrin; Kuipers, Oscar P.; Veening, Jan- Willem

    2011-01-01

    During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ. PMID:21841760

  15. Patenting medical diagnosis methods in Europe: Stanford University and time-lapse microscopy.

    PubMed

    Sterckx, Sigrid; Cockbain, Julian; Pennings, Guido

    2017-02-01

    In 2013, a European Patent for the technique of time-lapse microscopy was granted by the European Patent Office (EPO) to Stanford University and was subsequently opposed by Unisense FertiliTech A/S and by the European Society for Human Reproduction and Embryology (ESHRE), Sigrid Sterckx, Julian Cockbain and Guido Pennings. ESHRE et al.'s opposition was based on the argument that Stanford's patent was directed to a method of medical diagnosis, methods that are excluded from patentability by Article 53(c) of the European Patent Convention. The Opposition Division of the EPO rejected the oppositions in November 2015, and both opponents have now filed their appeals. In this paper, we comment on the Opposition Division decision and the grounds of appeal put forward by ESHRE et al.

  16. In vivo amyloid aggregation kinetics tracked by time-lapse confocal microscopy in real-time.

    PubMed

    Villar-Piqué, Anna; Espargaró, Alba; Ventura, Salvador; Sabate, Raimon

    2016-01-01

    Amyloid polymerization underlies an increasing number of human diseases. Despite this process having been studied extensively in vitro, aggregation is a difficult process to track in vivo due to methodological limitations and the slow kinetics of aggregation reactions in cells and tissues. Herein we exploit the amyloid properties of the inclusions bodies (IBs) formed by amyloidogenic proteins in bacteria to address the kinetics of in vivo amyloid aggregation. To this aim we used time-lapse confocal microscopy and a fusion of the amyloid-beta peptide (A β42) with a fluorescent reporter. This strategy allowed us to follow the intracellular kinetics of amyloid-like aggregation in real-time and to discriminate between variants exhibiting different in vivo aggregation propensity. Overall, the approach opens the possibility to assess the impact of point mutations as well as potential anti-aggregation drugs in the process of amyloid formation in living cells.

  17. Time-lapse microscopy and image analysis in basic and clinical embryo development research.

    PubMed

    Wong, C; Chen, A A; Behr, B; Shen, S

    2013-02-01

    Mammalian preimplantation embryo development is a complex process in which the exact timing and sequence of events are as essential as the accurate execution of the events themselves. Time-lapse microscopy (TLM) is an ideal tool to study this process since the ability to capture images over time provides a combination of morphological, dynamic and quantitative information about developmental events. Here, we systematically review the application of TLM in basic and clinical embryo research. We identified all relevant preimplantation embryo TLM studies published in English up to May 2012 using PubMed and Google Scholar. We then analysed the technical challenges involved in embryo TLM studies and how these challenges may be overcome with technological innovations. Finally, we reviewed the different types of TLM embryo studies, with a special focus on how TLM can benefit clinical assisted reproduction. Although new parameters predictive of embryo development potential may be discovered and used clinically to potentially increase the success rate of IVF, adopting TLM to routine clinical practice will require innovations in both optics and image analysis. Combined with such innovations, TLM may provide embryologists and clinicians with an important tool for making critical decisions in assisted reproduction. In this review, we perform a literature search of all published early embryo development studies that used time-lapse microscopy (TLM). From the literature, we discuss the benefits of TLM over traditional time-point analysis, as well as the technical difficulties and solutions involved in implementing TLM for embryo studies. We further discuss research that has successfully derived non-invasive markers that may increase the success rate of assisted reproductive technologies, primarily IVF. Most notably, we extend our discussion to highlight important considerations for the practical use of TLM in research and clinical settings.

  18. Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

    PubMed

    Lynch, Adam E; Triajianto, Junian; Routledge, Edwin

    2014-01-01

    Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

  19. Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

    PubMed Central

    Asada, Masahito; Goto, Yasuyuki; Yahata, Kazuhide; Yokoyama, Naoaki; Kawai, Satoru; Inoue, Noboru; Kaneko, Osamu; Kawazu, Shin-ichiro

    2012-01-01

    Background Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. Methodology/Principal Findings Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. Conclusions/Significance This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding. PMID:22506073

  20. 3D time-lapse seismic traveltime tomography for detecting near surface velocity variations: a case study from the Ketzin CO2 storage pilot site

    NASA Astrophysics Data System (ADS)

    Zhang, Fengjiao; Juhlin, Christopher; Huang, Fei; Lüth, Stefan

    2016-04-01

    Time-lapse seismic methods are an important tool for monitoring CO2 migration and storage in geological formations. Near surface variations are one of the major problems which may introduce time-lapse noise in the application of land based seismic monitoring. Conventional reflection seismic methods have difficulties in imaging near surface structures (10-30 m depth) due to the limitation of the methods themselves. Traveltime tomography is a commonly used method to reconstruct the subsurface velocity model. It can often provide extra information on near surface structures which is difficult to obtain by the conventional reflection seismic method. In this study, we apply traveltime tomography to 3D time-lapse seismic data sets acquired from at the Ketzin CO2 storage site. We also test different inversion strategies for traveltime tomography to investigate which one is more suitable for this case study. The results show good correlation with near surface variations obtained by other studies.

  1. Time-lapse microscopy studies of bystander effects induced by photosensitization

    NASA Astrophysics Data System (ADS)

    Chen, Yin-Chu; Redmond, Robert W.

    2006-02-01

    Reactive oxygen species (ROS) are involved in the pathogenesis of many critical diseases and are also utilized as cytotoxic agents in a variety of treatments for eradication of diseased tissue, including cancer. Oxidative stress ensues when the level of ROS in a system exceeds the antioxidant capacity. Oxidative stress can have local (direct) and long-range (bystander) effects in cells and tissue and this research was carried out to determine the spatial and temporal nature of the photosensitized bystander effect using time-lapse fluorescence microscopy. By initiating photosensitization in only a portion of the microscopic imaging field it was possible to differentiate direct from bystander effects in EMT-6 murine breast cancer cells in 6-well plates. Elevated ROS levels are seen immediately following photodynamic treatment in direct cells with a delayed increase in oxidative stress observed in bystander cells. Cytotoxicity is also seen at earlier times in direct cells and occurs in bystander cells in a delayed fashion. These studies confirm the existence of a bystander effect following photosensitization and implicate mediators capable of diffusing in an intercellular manner from directly photosensitized cells to bystander cells and also implicate increased oxidative stress as a mechanistic factor in generating damage in bystander cells.

  2. Quantitative comparison of multiframe data association techniques for particle tracking in time-lapse fluorescence microscopy.

    PubMed

    Smal, Ihor; Meijering, Erik

    2015-08-01

    Biological studies of intracellular dynamic processes commonly require motion analysis of large numbers of particles in live-cell time-lapse fluorescence microscopy imaging data. Many particle tracking methods have been developed in the past years as a first step toward fully automating this task and enabling high-throughput data processing. Two crucial aspects of any particle tracking method are the detection of relevant particles in the image frames and their linking or association from frame to frame to reconstruct the trajectories. The performance of detection techniques as well as specific combinations of detection and linking techniques for particle tracking have been extensively evaluated in recent studies. Comprehensive evaluations of linking techniques per se, on the other hand, are lacking in the literature. Here we present the results of a quantitative comparison of data association techniques for solving the linking problem in biological particle tracking applications. Nine multiframe and two more traditional two-frame techniques are evaluated as a function of the level of missing and spurious detections in various scenarios. The results indicate that linking techniques are generally more negatively affected by missing detections than by spurious detections. If misdetections can be avoided, there appears to be no need to use sophisticated multiframe linking techniques. However, in the practically likely case of imperfect detections, the latter are a safer choice. Our study provides users and developers with novel information to select the right linking technique for their applications, given a detection technique of known quality.

  3. Early events in insulin fibrillization studied by time-lapse atomic force microscopy.

    PubMed

    Podestà, Alessandro; Tiana, Guido; Milani, Paolo; Manno, Mauro

    2006-01-15

    The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation.

  4. A microfluidic system for long-term time-lapse microscopy studies of mycobacteria.

    PubMed

    Golchin, Solmaz A; Stratford, James; Curry, Richard J; McFadden, Johnjoe

    2012-11-01

    Phenotypic heterogeneity in bacterial populations is thought to contribute to a number of important phenomena including sporulation and persistence. The latter has clinical implications in many diseases such as tuberculosis, where persistence of Mycobacterium tuberculosis within the human host is believed to be the root cause of latent tuberculosis and the ability of a minority population of cells to survive antibiotic exposure, despite being genetically identical to the bulk population that are killed. However, phenotypic variations caused by non-genetic mechanisms are difficult to study because of the transient nature of the persistent state and thereby the requirement to observe individual cells in real-time. Recently, microfluidics, combined with time-lapse microscopy, has become a powerful tool for studying population heterogeneity in bacteria. However, growth and replication of mycobacterial cells provide particular problems for the development of microfluidic systems due to their tendency to grow in three dimensions. We here describe a novel microfluidic device for the observation of growth and antibiotic killing in individual mycobacterial cells. We constructed a microfluidic device suitable for studying single cell behavior in mycobacteria. The growth of single cells of Mycobacterium smegmatis expressing green fluorescent protein was monitored using a confocal laser scanning microscope. Within the device M. smegmatis cells were tightly confined within a hydrogel matrix thus promoting planar growth. Cell growth and killing was observed in the device with dead cells highlighted by uptake of propidium iodide. Conclusions/Significance. We demonstrate that our device allows real-time analysis and long-term culture of single cells of mycobacteria, and is able to support the study of cell death during the application of antibiotics. The device will allow observation of individual cells' cell genealogy to be determined and direct observation of rare states, such

  5. Quantitative time-lapse 3D seismic data interpretation from the pilot site of Ketzin (CO2 Storage): the level of noise

    NASA Astrophysics Data System (ADS)

    Ivanova, Alexandra; Lüth, Stefan; Kempka, Thomas

    2015-04-01

    The first European onshore pilot scale project for geological storage of carbon dioxide (CO2) was initiated in 2004 near Berlin (Germany). This project is multidisciplinary including 3D seismic time-lapse surveys as an essential tool for reservoir characterization at a depth of 650 m. A 3D pre-injection baseline seismic survey was acquired in 2005. CO2 injection into a sandstone saline aquifer started in 2008 and stopped in 2013 after 67 kilotons of CO2 had been injected. The 1st and 2nd 3D seismic repeat surveys were acquired after 22 and 61 kilotons of CO2 had been injected respectively. Time-lapse seismic processing, petrophysical data and geophysical logging of CO2 saturation levels have allowed for an estimate of the total amount of CO2 visible in the seismic data to be made. The close agreement (over 85%) between the injected and observed amount is encouraging for quantitative monitoring of a CO2 storage site using seismic methods. However this estimate contains a number of uncertainties. For example the most of the time delay values in the both 3D seismic repeat surveys within the amplitude anomaly are near the noise level of 1-2 ms, however a change of 1 ms in the time delay affects significantly the mass estimate, thus the choice of the time-delay cutoff is crucial. In this study we put bounds into the noise in seismic data from Ketzin using results of 3D flow simulations.

  6. Presentation of a High Resolution Time Lapse 3D Groundwater Model of Metsähovi for Calculating the Gravity Effect of Groundwater in Local Scale

    NASA Astrophysics Data System (ADS)

    Hokkanen, T. M.; Hartikainen, A.; Raja-Halli, A.; Virtanen, H.; Makinen, J.

    2015-12-01

    INTRODUCTION The aim of this study is to construct a fine resolution time lapse groundwater (GW) model of Metsähovi (MH). GW, geological, and soil moisture (SM) data were collected for several years to achieve the goal. The knowledge of the behavior of the GW at local scale is essential for superconductive gravimeter (SG) investigations performing in MH. DESCRIPTION OF THE DATA Almost 50 sensors have been recorded SM data some 6 years with 1 to 5 minutes sampling frequency. The GW table has been monitored, both in bedrock and in soil, in many stages with all together 15 piezometers. Two geological sampling campaigns were conducted to get the knowledge of hydrological properties of soil in the study area of 200×200 m2 around SG station in MH. PRINCIPLE OF TIME LAPSE 3D HYDROGEOLOGICAL MODEL The model of study site consists of the surfaces of ground and bedrock gridded with 2×2 m2 resolution. The height of GW table was interpolated to 2×2×0.1 m3 grid between GW and SM monitoring points. Close to the outline of the study site and areas lacking of sensors GW table was defined by extrapolation and considering the geological information of the area. The bedrock porosity is 2% and soil porosity determined by geological information and SM recordings is from 5 to 35%. Only fully saturated media is considered in the time lapse model excluding unsaturated one. BENEFICIERS With a new model the fluctuation of GW table can be followed with ranging time lapses from 1 minute to 1 month. The gravity effect caused by the variation of GW table can be calculated more accurate than before in MH. Moreover, the new model can be validated and refined by measured gravity, i.e. hydrological model can be improved by SG recordings (Figure 1).

  7. Rapid and retrievable recording of big data of time-lapse 3D shadow images of microbial colonies.

    PubMed

    Ogawa, Hiroyuki; Nasu, Senshi; Takeshige, Motomu; Saito, Mikako; Matsuoka, Hideaki

    2015-05-15

    We formerly developed an automatic colony count system based on the time-lapse shadow image analysis (TSIA). Here this system has been upgraded and applied to practical rapid decision. A microbial sample was spread on/in an agar plate with 90 mm in diameter as homogeneously as possible. We could obtain the results with several strains that most of colonies appeared within a limited time span. Consequently the number of colonies reached a steady level (Nstdy) and then unchanged until the end of long culture time to give the confirmed value (Nconf). The equivalence of Nstdy and Nconf as well as the difference of times for Nstdy and Nconf determinations were statistically significant at p < 0.001. Nstdy meets the requirement of practical routines treating a large number of plates. The difference of Nstdy and Nconf, if any, may be elucidated by means of retrievable big data. Therefore Nconf is valid for official documentation.

  8. Segmentation Method of Time-Lapse Microscopy Images with the Focus on Biocompatibility Assessment.

    PubMed

    Soukup, Jindřich; Císař, Petr; Šroubek, Filip

    2016-06-01

    Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.

  9. Realtime infiltration process monitoring in macroporous soil - a plot-scale experiment accompanied by high-resolution time-lapse 3D GPR

    NASA Astrophysics Data System (ADS)

    Jackisch, Conrad; Allroggen, Niklas

    2016-04-01

    Infiltration and quick vertical redistribution of event water through rapid subsurface flow in soil structures is one of the key issues in hydrology. Although the importance of preferential flow is broadly recognised, our theories, observation techniques and modelling approaches lose grounds when the assumption of well-mixed states in REVs collapses. To characterise the combination of advective and diffusive flow is especially challenging. We have shown in earlier studies that a combination of TDR monitoring, dye- and salt-tracer recovery and time-lapse 3D GPR in irrigation experiments provides means to characterise infiltration dynamics at the plot- and hillslope-scale also in highly structured soils. We pinpointed that the spatial and temporal resolution requires special attention and improvement - particularly owing to the facts of high velocity (10-3 ms-1) of advective flow and small scale (10-2 m) of the respective flow structures. We present insights from a novel technique of continuous high-resolution time-lapse 3D GPR measurements during and after a plot-scale (1 m x 1 m) irrigation experiment. Continuous TDR soil moisture measurements, dye tracer excavation and salt-tracer samples are used as qualitative and quantitative references. While classical infiltration experiments either look at spatial patterns or temporal dynamics at singular gauges, we highlight the advantage of combining both to achieve a more complete image of the infiltration process. Although operating at the limits of the techniques this setup enables non-invasive observation of preferential flow processes in the field and allows to explore and characterise macropore matrix exchange.

  10. Time-lapse ultrashort pulse microscopy of infection in three-dimensional versus two-dimensional culture environments reveals enhanced extra-chromosomal virus replication compartment formation

    NASA Astrophysics Data System (ADS)

    Gibbs, Holly C.; Sing, Garwin; Armas, Juan Carlos González; Campbell, Colin J.; Ghazal, Peter; Yeh, Alvin T.

    2013-03-01

    The mechanisms that enable viruses to harness cellular machinery for their own survival are primarily studied in cell lines cultured in two-dimensional (2-D) environments. However, there are increasing reports of biological differences between cells cultured in 2-D versus three-dimensional (3-D) environments. Here we report differences in host-virus interactions based on differences in culture environment. Using ultrashort pulse microscopy (UPM), a form of two-photon microscopy that utilizes sub-10-fs pulses to efficiently excite fluorophores, we have shown that de novo development of extra-chromosomal virus replication compartments (VRCs) upon murine cytomegalovirus (mCMV) infection is markedly enhanced when host cells are cultured in 3-D collagen gels versus 2-D monolayers. In addition, time-lapse imaging revealed that mCMV-induced VRCs have the capacity to grow by coalescence. This work supports the future potential of 3-D culture as a useful bridge between traditional monolayer cultures and animal models to study host-virus interactions in a more physiologically relevant environment for the development of effective anti-viral therapeutics. These advances will require broader adoption of modalities, such as UPM, to image deep within scattering tissues.

  11. Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

    PubMed

    Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

    2016-07-01

    Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events.

  12. Pixel timing correction in time-lapsed calcium imaging using point scanning microscopy.

    PubMed

    Boiroux, Dimitri; Oke, Yoshihiko; Miwakeichi, Fumikazu; Oku, Yoshitaka

    2014-11-30

    In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.

  13. Automated segmentation and tracking of non-rigid objects in time-lapse microscopy videos of polymorphonuclear neutrophils.

    PubMed

    Brandes, Susanne; Mokhtari, Zeinab; Essig, Fabian; Hünniger, Kerstin; Kurzai, Oliver; Figge, Marc Thilo

    2015-02-01

    Time-lapse microscopy is an important technique to study the dynamics of various biological processes. The labor-intensive manual analysis of microscopy videos is increasingly replaced by automated segmentation and tracking methods. These methods are often limited to certain cell morphologies and/or cell stainings. In this paper, we present an automated segmentation and tracking framework that does not have these restrictions. In particular, our framework handles highly variable cell shapes and does not rely on any cell stainings. Our segmentation approach is based on a combination of spatial and temporal image variations to detect moving cells in microscopy videos. This method yields a sensitivity of 99% and a precision of 95% in object detection. The tracking of cells consists of different steps, starting from single-cell tracking based on a nearest-neighbor-approach, detection of cell-cell interactions and splitting of cell clusters, and finally combining tracklets using methods from graph theory. The segmentation and tracking framework was applied to synthetic as well as experimental datasets with varying cell densities implying different numbers of cell-cell interactions. We established a validation framework to measure the performance of our tracking technique. The cell tracking accuracy was found to be >99% for all datasets indicating a high accuracy for connecting the detected cells between different time points.

  14. Time-lapse contact microscopy of cell cultures based on non-coherent illumination

    NASA Astrophysics Data System (ADS)

    Gabriel, Marion; Balle, Dorothée; Bigault, Stéphanie; Pornin, Cyrille; Gétin, Stéphane; Perraut, François; Block, Marc R.; Chatelain, François; Picollet-D'Hahan, Nathalie; Gidrol, Xavier; Haguet, Vincent

    2015-10-01

    Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.

  15. Time-lapse contact microscopy of cell cultures based on non-coherent illumination

    PubMed Central

    Gabriel, Marion; Balle, Dorothée; Bigault, Stéphanie; Pornin, Cyrille; Gétin, Stéphane; Perraut, François; Block, Marc R.; Chatelain, François; Picollet-D’hahan, Nathalie; Gidrol, Xavier; Haguet, Vincent

    2015-01-01

    Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell. PMID:26459014

  16. Time-Lapse, in Situ Imaging of Ice Crystal Growth Using Confocal Microscopy

    PubMed Central

    2016-01-01

    Ice crystals nucleate and grow when a water solution is cooled below its freezing point. The growth velocities and morphologies of the ice crystals depend on many parameters, such as the temperature of ice growth, the melting temperature, and the interactions of solutes with the growing crystals. Three types of morphologies may appear: dendritic, cellular (or fingerlike), or the faceted equilibrium form. Understanding and controlling which type of morphology is formed is essential in several domains, from biology to geophysics and materials science. Obtaining, in situ, three dimensional observations without introducing artifacts due to the experimental technique is nevertheless challenging. Here we show how we can use laser scanning confocal microscopy to follow in real-time the growth of smoothed and faceted ice crystals in zirconium acetate solutions. Both qualitative and quantitative observations can be made. In particular, we can precisely measure the lateral growth velocity of the crystals, a measure otherwise difficult to obtain. Such observations should help us understand the influence of the parameters that control the growth of ice crystals in various systems. PMID:27917410

  17. Automated tracking of temporal displacements of a red blood cell obtained by time-lapse digital holographic microscopy.

    PubMed

    Moon, Inkyu; Yi, Faliu; Rappaz, Benjamin

    2016-01-20

    Red blood cell (RBC) phase images that are numerically reconstructed by digital holographic microscopy (DHM) can describe the cell structure and dynamics information beneficial for a quantitative analysis of RBCs. However, RBCs investigated with time-lapse DHM undergo temporal displacements when their membranes are loosely attached to the substrate during sedimentation on a glass surface or due to the microscope drift. Therefore, we need to develop a tracking algorithm to localize the same RBC among RBC image sequences and dynamically monitor its biophysical cell parameters; this information is helpful for studies on RBC-related diseases and drug tests. Here, we propose a method, which is a combination of the mean-shift algorithm and Kalman filter, to track a single RBC and demonstrate that the optical path length of the single RBC can be continually extracted from the tracked RBC. The Kalman filter is utilized to predict the target RBC position in the next frame. Then, the mean-shift algorithm starts execution from the predicted location, and a robust kernel, which is adaptive to changes in the RBC scale, shape, and direction, is designed to improve the accuracy of the tracking. Finally, the tracked RBC is segmented and parameters such as the RBC location are extracted to update the Kalman filter and the kernel function for mean-shift tracking; the characteristics of the target RBC are dynamically observed. Experimental results show the feasibility of the proposed algorithm.

  18. In vivo time-lapse imaging of skin burn wound healing using second-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Yasui, Takeshi; Tanaka, Ryosuke; Hase, Eiji; Fukushima, Shu-ichiro; Araki, Tsutomu

    2014-02-01

    Wound healing is a process to repair the damaged tissue caused by thermal burn, incised wound, or stab wound. Although the wound healing has many aspects, it is common for dynamics of collagen fiber, such as decomposition, production, or growth, to be closely related with wound healing. If such the healing process can be visualized as a timelapse image of the collagen fiber in the same subject, one may obtain new findings regarding biological repairing mechanisms in the healing process. In this article, to investigate the temporal modoification of dermal collagen fiber in the burn wound healing, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, minimal invasiveness, deep penetration, the absence of interference from background light, and in vivo measurement without additional staining. Since SHG light arises from a non-centrosymmetric triple helix of three polypeptide chains in the collagen molecule, SHG intensity sensitively reflects the structure maturity of collagen molecule and its aggregates. A series of time-lapse SHG images during the wound healing process of 2 weeks clearly indicated that condensation and melting of dermal collagen fibers by the deep dermal burn, decomposition of the damaged collagen fibers in the inflammation phase, production of new collagen fibers in the proliferation phase, and the growth of the new collagen fibers in the remodeling phase. These results show a high potential of SHG microscopy for optical assessment of the wound healing process in vivo.

  19. Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy.

    PubMed Central

    Schoenenberger, C A; Hoh, J H

    1994-01-01

    We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine Kidney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological conditions, and time-lapse movies representing approximately 60 s real time per frame were assembled. In normal MDCK cells, two types of protrusions in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges formed transiently over the time scale of minutes to tens of minutes. Second, spike-like protrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenic transformant derived from MDCK cells, grows very flat on glass. During AFM imaging, these cells sometimes round up and detach from the substrate. In light microscopic observations of parallel preparations, cells rarely detach, suggesting that this is an active response of these cells to irritation by the AFM tip. R5 cells often extend processes that are supported by actin stress fibers. During imaging with the AFM, these processes withdraw at a rate of 1-5 microns/min, similar to that observed by light microscopy. During the withdrawal, movement of the stress fibers can be clearly seen. In the flat periphery of these cells, the transport of intracellular particles along cytoskeletal elements was seen. In addition, we have observed two types of wave-like movements through the cell, which appear to be an organized rearrangement of cytoplasm. One type of wave moves radially out from center of the cell while the other moves circularly along the cell periphery. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7948707

  20. 3D structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Dougherty, William M.; Goodwin, Paul C.

    2011-03-01

    Three-dimensional structured illumination microscopy achieves double the lateral and axial resolution of wide-field microscopy, using conventional fluorescent dyes, proteins and sample preparation techniques. A three-dimensional interference-fringe pattern excites the fluorescence, filling in the "missing cone" of the wide field optical transfer function, thereby enabling axial (z) discrimination. The pattern acts as a spatial carrier frequency that mixes with the higher spatial frequency components of the image, which usually succumb to the diffraction limit. The fluorescence image encodes the high frequency content as a down-mixed, moiré-like pattern. A series of images is required, wherein the 3D pattern is shifted and rotated, providing down-mixed data for a system of linear equations. Super-resolution is obtained by solving these equations. The speed with which the image series can be obtained can be a problem for the microscopy of living cells. Challenges include pattern-switching speeds, optical efficiency, wavefront quality and fringe contrast, fringe pitch optimization, and polarization issues. We will review some recent developments in 3D-SIM hardware with the goal of super-resolved z-stacks of motile cells.

  1. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  2. AMS Time Lapse Installation

    NASA Video Gallery

    A time lapse video compilation of the installation of the Alpha Magnetic Spectrometer on the International Space Station’s starboard truss using the station’s robotic arm, Canadarm2, during the...

  3. Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.

    PubMed

    Gfrerer, Lisa; Dougherty, Max; Liao, Eric C

    2013-09-30

    Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

  4. Time-lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation.

    PubMed

    Seiler, Christof; Gazdhar, Amiq; Reyes, Mauricio; Benneker, Lorin M; Geiser, Thomas; Siebenrock, Klaus A; Gantenbein-Ritter, Benjamin

    2014-09-01

    Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high-density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time-lapse microscopy to record cell shape, using phase-contrast microscopy. The cells were segmented using a custom-made image-processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven-dimensional feature vectors, using a tree-like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time-lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non-invasive automated time-lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time-points. The classification is challenged by cell density, proliferation and possible unknown donor-specific factors, which affect the performance of

  5. Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with real-time dosimetry

    PubMed Central

    Orzekowsky-Schroeder, Regina; Klinger, Antje; Freidank, Sebastian; Linz, Norbert; Eckert, Sebastian; Hüttmann, Gereon; Gebert, Andreas; Vogel, Alfred

    2014-01-01

    Gut mucosa is an important interface between body and environment. Immune response and healing processes of murine small intestinal mucosa were investigated by intravital time-lapse two-photon excited autofluorescence microscopy of the response to localized laser-induced damage. Epithelial lesions were created by 355-nm, 500-ps pulses from a microchip laser that produced minute cavitation bubbles. Size and dynamics of these bubbles were monitored using a novel interferometric backscattering technique with 80 nm resolution. Small bubbles (< 2.5 µm maximum radius) merely resulted in autofluorescence loss of the target cell. Larger bubbles (7-25 µm) affected several cells and provoked immigration of immune cells (polymorphonuclear leucocytes). Damaged cells were expelled into the lumen, and the epithelium healed within 2 hours by stretching and migration of adjacent epithelial cells. PMID:25360369

  6. Atomic resolution 3D electron diffraction microscopy

    SciTech Connect

    Miao, Jianwei; Ohsuna, Tetsu; Terasaki, Osamu; O'Keefe, Michael A.

    2002-03-01

    Electron lens aberration is the major barrier limiting the resolution of electron microscopy. Here we describe a novel form of electron microscopy to overcome electron lens aberration. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a 2 x 2 x 2 unit cell nano-crystal (framework of LTA [Al12Si12O48]8) can be ab initio determined at the resolution of 1 Angstrom from a series of simulated noisy diffraction pattern projections with rotation angles ranging from -70 degrees to +70 degrees in 5 degrees increments along a single rotation axis. This form of microscopy (which we call 3D electron diffraction microscopy) does not require any reference waves, and can image the 3D structure of nanocrystals, as well as non-crystalline biological and materials science samples, with the resolution limited only by the quality of sample diffraction.

  7. In-vivo Optical Tomography of Small Scattering Specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster

    PubMed Central

    Arranz, Alicia; Dong, Di; Zhu, Shouping; Savakis, Charalambos; Tian, Jie; Ripoll, Jorge

    2014-01-01

    Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window - thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously. PMID:25471694

  8. An automated system for collection of time-lapse 3D radar data to investigate vadose zone flow and transport processes

    NASA Astrophysics Data System (ADS)

    Mangel, A. R.; Moysey, S. M.

    2013-12-01

    Capturing three-dimensional ground-penetrating radar (GPR) images can significantly enhance our understanding of subsurface variability during vadose zone flow and transport processes. The high spatial sampling (i.e., small step sizes between profiles) required to collect full resolution 3D data can be a major challenge - particularly for high frequency imaging of detailed structures such as those related to preferential flow patterns in soils. We have developed an automated system for collecting GPR data to address these challenges. The system is based on the Sensors and Software SPIDAR (OEM NIC) platform running a 1000MHz source and receiver antenna that can be independently positioned using a 2-axis motion control system, with both the radar and positioning components integrated through LabView. The antennas can be positioned independently along a rail parallel with the x-axis, which can itself be moved along a second set of rails along the y-axis. The positioning accuracy along each axis has been estimated to be 3um and 0.2mm along each direction, respectively, thus indicating that high resolution positioning for accurate 3D imaging is readily attained. The integrated radar and positioning system is currently capable of collecting up to 100 traces per second over a 25ns time window with 4 stacks, or an equivalent lateral velocity of approximately 50cm/s with traces collected every 0.5cm along the profile. This high speed data collection means that a full 3D section of data (>75,000 traces) over a 0.75m x 1.5m area can be collected in under 20 minutes at sub-centimeter resolution, implying that near real-time imaging of infiltration over reasonably large areas can be achieved. In our case, the radar system has been implemented for a lab environment where it is able to perform imaging experiments over a 4m x 4m x 2m (LxWxH) sand-filled tank. In this presentation we will provide examples of three dimensional data collected over the tank. Experiments imaging rocks

  9. In-vivo optical tomography of small scattering specimens: time-lapse 3D imaging of the head eversion process in Drosophila melanogaster.

    PubMed

    Arranz, Alicia; Dong, Di; Zhu, Shouping; Savakis, Charalambos; Tian, Jie; Ripoll, Jorge

    2014-12-04

    Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window--thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.

  10. Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

    PubMed

    Farnum, C E; Turgai, J; Wilsman, N J

    1990-09-01

    The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

  11. A semi-Markov model for mitosis segmentation in time-lapse phase contrast microscopy image sequences of stem cell populations.

    PubMed

    Liu, An-An; Li, Kang; Kanade, Takeo

    2012-02-01

    We propose a semi-Markov model trained in a max-margin learning framework for mitosis event segmentation in large-scale time-lapse phase contrast microscopy image sequences of stem cell populations. Our method consists of three steps. First, we apply a constrained optimization based microscopy image segmentation method that exploits phase contrast optics to extract candidate subsequences in the input image sequence that contains mitosis events. Then, we apply a max-margin hidden conditional random field (MM-HCRF) classifier learned from human-annotated mitotic and nonmitotic sequences to classify each candidate subsequence as a mitosis or not. Finally, a max-margin semi-Markov model (MM-SMM) trained on manually-segmented mitotic sequences is utilized to reinforce the mitosis classification results, and to further segment each mitosis into four predefined temporal stages. The proposed method outperforms the event-detection CRF model recently reported by Huh as well as several other competing methods in very challenging image sequences of multipolar-shaped C3H10T1/2 mesenchymal stem cells. For mitosis detection, an overall precision of 95.8% and a recall of 88.1% were achieved. For mitosis segmentation, the mean and standard deviation for the localization errors of the start and end points of all mitosis stages were well below 1 and 2 frames, respectively. In particular, an overall temporal location error of 0.73 ± 1.29 frames was achieved for locating daughter cell birth events.

  12. 3D Viscoelastic traction force microscopy.

    PubMed

    Toyjanova, Jennet; Hannen, Erin; Bar-Kochba, Eyal; Darling, Eric M; Henann, David L; Franck, Christian

    2014-10-28

    Native cell-material interactions occur on materials differing in their structural composition, chemistry, and physical compliance. While the last two decades have shown the importance of traction forces during cell-material interactions, they have been almost exclusively presented on purely elastic in vitro materials. Yet, most bodily tissue materials exhibit some level of viscoelasticity, which could play an important role in how cells sense and transduce tractions. To expand the realm of cell traction measurements and to encompass all materials from elastic to viscoelastic, this paper presents a general, and comprehensive approach for quantifying 3D cell tractions in viscoelastic materials. This methodology includes the experimental characterization of the time-dependent material properties for any viscoelastic material with the subsequent mathematical implementation of the determined material model into a 3D traction force microscopy (3D TFM) framework. Utilizing this new 3D viscoelastic TFM (3D VTFM) approach, we quantify the influence of viscosity on the overall material traction calculations and quantify the error associated with omitting time-dependent material effects, as is the case for all other TFM formulations. We anticipate that the 3D VTFM technique will open up new avenues of cell-material investigations on even more physiologically relevant time-dependent materials including collagen and fibrin gels.

  13. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  14. Diffusion of solutes inside bacterial colonies immobilized in model cheese depends on their physicochemical properties: a time-lapse microscopy study

    PubMed Central

    Floury, Juliane; El Mourdi, Ilham; Silva, Juliana V. C.; Lortal, Sylvie; Thierry, Anne; Jeanson, Sophie

    2015-01-01

    During cheese processing and ripening, bacteria develop as colonies. Substrates and metabolites must then diffuse either from or into the colonies. Exploring how the inner cells of the colony access the substrates or get rid of the products leads to study the diffusion of solutes inside bacterial colonies immobilized in cheese. Diffusion limitations of substrates within the bacterial colony could lead to starvation for the cells in the center of the colony. This study aimed at better understands ripening at the colony level, by investigating how diffusion phenomena inside colonies vary depending on both the physicochemical properties of the solutes and Lactococcus lactis strain. Dextrans (4, 70, and 155 kDa) and milk proteins (BSA, lactoferrin and αS1-casein) of different sizes and physicochemical properties were chosen as model of diffusing solutes, and two L. lactis strains presenting different surface properties were immobilized as colonies in a model cheese. Diffusion of solutes inside and around colonies was experimentally followed by time-lapse confocal microscopy. Dextran solutes diffused inside both lactococci colonies with a non-significantly different effective diffusion coefficient, which depended mainly on size of the solute. However, whereas flexible and neutral hydrophilic polymers such as dextran can diffuse inside colonies whatever its size, none of the three proteins investigated in this study could penetrate inside lactococci colonies. Therefore, the diffusion behavior of macromolecules through bacterial colonies immobilized in a model cheese did not only depends on the size of the diffusing solutes, but also and mainly on their physicochemical properties. Milk caseins are probably first hydrolyzed by the cell wall proteases of L. lactis and/or other proteases present in the cheese, and then the generated peptides diffuse inside colonies to be further metabolized into smaller peptides and amino acids by all the cells located inside the colonies

  15. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    NASA Astrophysics Data System (ADS)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  16. A-3 Construction Time Lapse

    NASA Technical Reports Server (NTRS)

    2009-01-01

    A time lapse from start to finish of steel erection for the 235-foot tall A-3 Test Stand. Ground work for the stand was broken in August 2008 and the final structural steel beam was placed April 9, 2009.

  17. Simple buffers for 3D STORM microscopy.

    PubMed

    Olivier, Nicolas; Keller, Debora; Rajan, Vinoth Sundar; Gönczy, Pierre; Manley, Suliana

    2013-06-01

    3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

  18. 3D differential phase contrast microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-03-01

    We demonstrate three-dimensional (3D) optical phase and amplitude reconstruction based on coded source illumination using a programmable LED array. Multiple stacks of images along the optical axis are computed from recorded intensities captured by multiple images under off-axis illumination. Based on the first Born approximation, a linear differential phase contrast (DPC) model is built between 3D complex index of refraction and the intensity stacks. Therefore, 3D volume reconstruction can be achieved via a fast inversion method, without the intermediate 2D phase retrieval step. Our system employs spatially partially coherent illumination, so the transverse resolution achieves twice the NA of coherent systems, while axial resolution is also improved 2× as compared to holographic imaging.

  19. 3D differential phase contrast microscopy

    PubMed Central

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-01-01

    We demonstrate 3D phase and absorption recovery from partially coherent intensity images captured with a programmable LED array source. Images are captured through-focus with four different illumination patterns. Using first Born and weak object approximations (WOA), a linear 3D differential phase contrast (DPC) model is derived. The partially coherent transfer functions relate the sample’s complex refractive index distribution to intensity measurements at varying defocus. Volumetric reconstruction is achieved by a global FFT-based method, without an intermediate 2D phase retrieval step. Because the illumination is spatially partially coherent, the transverse resolution of the reconstructed field achieves twice the NA of coherent systems and improved axial resolution. PMID:27867705

  20. Live Cells as Dynamic Laboratories: Time Lapse Raman Spectral Microscopy of Nanoparticles with Both IgE Targeting and pH-Sensing Functions

    DOE PAGES

    Nowak-Lovato, Kristy L.; Rector, Kirk D.

    2012-01-01

    Tmore » his review captures the use of live cells as dynamic microlaboratories through implementation of labeled nanoparticles (nanosensors) that have both sensing and targeting functions.he addition of 2,4-ε-dinitrophenol-L-lysine (DNP) as a FcεRI targeting ligand and 4-mercaptopyridine (4-MPy) as a pH-sensing ligand enables spatial and temporal monitoring of FcεRI receptors and their pH environment within the endocytic pathway.o ensure reliability, the sensor is calibrated in vivo using the ionophore nigericin and standard buffer solutions to equilibrate the external [ H + ] concentration with that of the cell compartments.his review highlights the nanosensors, ability to traffic and respond to pH of receptor-bound nanosensors (1) at physiological temperature ( 37 ° C ) versus room temperature ( 25 ° C ) , (2) after pharmacological treatment with bafilomycin, an H + ATPase pump inhibitor, or amiloride, an inhibitor of Na + / H + exchange, and (3) in response to both temperature and pharmacological treatment. Whole-cell, time lapse images are demonstrated to show the ability to transform live cells into dynamic laboratories to monitor temporal and spatial endosomal pH.he versatility of these probes shows promise for future applications relevant to intracellular trafficking and intelligent drug design.« less

  1. Live Cells as Dynamic Laboratories: Time Lapse Raman Spectral Microscopy of Nanoparticles with Both IgE Targeting and pH-Sensing Functions

    PubMed Central

    Nowak-Lovato, Kristy L.; Rector, Kirk D.

    2012-01-01

    This review captures the use of live cells as dynamic microlaboratories through implementation of labeled nanoparticles (nanosensors) that have both sensing and targeting functions. The addition of 2,4-ε-dinitrophenol-L-lysine (DNP) as a FcεRI targeting ligand and 4-mercaptopyridine (4-MPy) as a pH-sensing ligand enables spatial and temporal monitoring of FcεRI receptors and their pH environment within the endocytic pathway. To ensure reliability, the sensor is calibrated in vivo using the ionophore nigericin and standard buffer solutions to equilibrate the external [H+] concentration with that of the cell compartments. This review highlights the nanosensors, ability to traffic and respond to pH of receptor-bound nanosensors (1) at physiological temperature (37°C) versus room temperature (25°C), (2) after pharmacological treatment with bafilomycin, an H+ ATPase pump inhibitor, or amiloride, an inhibitor of Na+/H+ exchange, and (3) in response to both temperature and pharmacological treatment. Whole-cell, time lapse images are demonstrated to show the ability to transform live cells into dynamic laboratories to monitor temporal and spatial endosomal pH. The versatility of these probes shows promise for future applications relevant to intracellular trafficking and intelligent drug design. PMID:22778738

  2. A one-piece 3D printed flexure translation stage for open-source microscopy

    NASA Astrophysics Data System (ADS)

    Sharkey, James P.; Foo, Darryl C. W.; Kabla, Alexandre; Baumberg, Jeremy J.; Bowman, Richard W.

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  3. Frames-Based Denoising in 3D Confocal Microscopy Imaging.

    PubMed

    Konstantinidis, Ioannis; Santamaria-Pang, Alberto; Kakadiaris, Ioannis

    2005-01-01

    In this paper, we propose a novel denoising method for 3D confocal microscopy data based on robust edge detection. Our approach relies on the construction of a non-separable frame system in 3D that incorporates the Sobel operator in dual spatial directions. This multidirectional set of digital filters is capable of robustly detecting edge information by ensemble thresholding of the filtered data. We demonstrate the application of our method to both synthetic and real confocal microscopy data by comparing it to denoising methods based on separable 3D wavelets and 3D median filtering, and report very encouraging results.

  4. Microscopy in 3D: a biologist’s toolbox

    PubMed Central

    Fischer, Robert S.; Wu, Yicong; Kanchanawong, Pakorn; Shroff, Hari; Waterman, Clare M.

    2012-01-01

    The power of fluorescence microscopy to study cellular structures and macromolecular complexes spans a wide range of size scales, from studies of cell behavior and function in physiological, three-dimensional (3D) environments, to understanding the molecular architecture of organelles. At each length scale, the challenge in 3D imaging is to extract the most spatial and temporal resolution possible while limiting photodamage/bleaching to living cells. A number of advancements in 3D fluorescence microscopy now offer higher resolution, improved speed, and reduced photobleaching relative to traditional point-scanning microscopy methods. Here, we discuss a few specific microscopy modalities that we believe will be particularly advantageous in imaging cells and subcellular structures in physiologically relevant 3D environments. PMID:22047760

  5. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  6. 3D microscopy - new powerful tools in geomaterials characterization

    NASA Astrophysics Data System (ADS)

    Mauko Pranjić, Alenka; Mladenovič, Ana; Turk, Janez; Šajna, Aljoša; Čretnik, Janko

    2016-04-01

    Microtomography (microCT) is becoming more and more widely recognized in geological sciences as a powerful tool for the spatial characterization of rock and other geological materials. Together with 3D image analysis and other complementary techniques, it has the characteristics of an innovative and non-destructive 3D microscopical technique. On the other hand its main disadvantages are low availability (only a few geological laboratories are equipped with high resolution tomographs), the relatively high prices of testing connected with the use of an xray source, technical limitations connected to the resolution and imaging of certain materials, as well as timeconsuming and complex 3D image analysis, necessary for quantification of 3D tomographic data sets. In this work three examples are presented of optimal 3D microscopy analysis of geomaterials in construction such as porosity characterization of impregnated sandstone, aerated concrete and marble prone to bowing. Studies include processes of microCT imaging, 3D data analysis and fitting of data with complementary analysis, such as confocal microscopy, mercury porosimetry, gas sorption, optical/fluorescent microscopy and scanning electron microscopy. Present work has been done in the frame of national research project 3D and 4D microscopy development of new powerful tools in geosciences (ARRS J1-7148) funded by Slovenian Research Agency.

  7. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  8. Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy.

    PubMed

    Tanaka, Aya; Tanaka, Ryosuke; Kasai, Nahoko; Tsukada, Shingo; Okajima, Takaharu; Sumitomo, Koji

    2015-07-01

    Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes.

  9. Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy

    PubMed Central

    Mulligan, Jeffrey A.; Bordeleau, François; Reinhart-King, Cynthia A.; Adie, Steven G.

    2017-01-01

    Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research. PMID:28271010

  10. A method to investigate radial glia cell behavior using two-photon time-lapse microscopy in an ex vivo model of spinal cord development

    PubMed Central

    Pakan, Janelle M. P.; McDermott, Kieran W.

    2014-01-01

    The mammalian central nervous system (CNS) develops from multipotent progenitor cells, which proliferate and differentiate into the various cell types of the brain and spinal cord. Despite the wealth of knowledge from progenitor cell culture studies, there is a significant lack of understanding regarding dynamic progenitor cell behavior over the course of development. This is in part due to shortcomings in the techniques available to study these processes in living tissues as they are occurring. In order to investigate cell behavior under physiologically relevant conditions we established an ex vivo model of the developing rat spinal cord. This method allows us to directly observe specific populations of cells ex vivo in real time and over extended developmental periods as they undergo proliferation, migration, and differentiation in the CNS. Previous investigations of progenitor cell behavior have been limited in either spatial or temporal resolution (or both) due to the necessity of preserving tissue viability and avoiding phototoxic effects of fluorescent imaging. The method described here overcomes these obstacles. Using two-photon and confocal microscopy and transfected organotypic spinal cord slice cultures we have undertaken detailed imaging of a unique population of neural progenitors, radial glial cells. This method uniquely enables analysis of large populations as well as individual cells; ultimately resulting in a 4D dataset of progenitor cell behavior for up to 7 days during embryonic development. This approach can be adapted to study a variety of cell populations at different stages of development using appropriate promoter driven fluorescent protein expression. The ability to control the tissue micro-environment makes this ex vivo method a powerful tool to elucidate the underlying molecular mechanisms regulating cell behavior during embryonic development. PMID:24782718

  11. Time-lapse atomic force microscopy observations of the morphology, growth rate, and spontaneous alignment of nanofibers containing a peptide-amphiphile from the hepatitis G virus (NS3 protein).

    PubMed

    Weroński, Konrad J; Cea, Pilar; Diez-Peréz, Ismael; Busquets, Maria Antonia; Prat, Josefina; Girona, Victoria

    2010-01-14

    Time-lapse atomic force microscopy is used in this contribution to directly watch the growth of nanofibers of a lipidated peptide on a mica surface. Specifically, the studied lipopeptide is the palmitoyl derivative of the fragment 505-514 of NS3 protein from the hepatitis G virus, abbreviated as Palmitoyl-NS3 (505-514). Data on the morphology, growth rate, and orientation of these peptide-amphiphile nanofibers have been obtained. From these data, it can be concluded that this synthetic lipopeptide forms two types of fiber-like aggregates: (i) half-spherical fibrous aggregates with lengths of hundreds of nanometers and (ii) spherical fibrous aggregates with lengths of several micrometers. In addition, when a fresh lipopeptide aqueous solution is deposited onto a mica surface, the aggregates spontaneously orient parallel to each other, yielding well-aligned nanofibers on large areas of the mica surface. A significant growth in both the length and the number of the fibers was observed during the first minutes after the solution deposition. Elongation of the fibrous aggregates from one end is more frequent, though elongation from both ends also occurs, with growth rates in the 4-5 nm/s range. The effects of dilution, mechanical perturbation, and pH on the aggregation behavior of Palmitoyl-NS3 (505-514) are also detailed in this paper.

  12. 3D super-resolution microscopy of bacterial division machinery

    NASA Astrophysics Data System (ADS)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.

    2016-08-01

    Super-resolution microscopy is a promising tool for the field of microbiology, as bacteria sizes are comparable to the resolution limit of light microscopy. Bacterial division machinery and FtsZ protein in particular attract much attention of scientists who use different super-resolution microscopy techniques, but most of the available data on FtsZ structures was obtained using two-dimensional (2D) super-resolution microscopy. Using 3D single-molecule localization microscopy (SMLM, namely dSTORM) to visualize FtsZ, we demonstrate that this approach allows more accurate interpretation of super-resolution images and provides new opportunities for the study of complex structures like bacterial divisome.

  13. Rim Fire Time Lapse, August 2013

    NASA Video Gallery

    Time-lapse photography shows various perspectives of the 2013 Rim Fire, as viewed from Yosemite National Park. The first part of this video is from the Crane Flat Helibase. The fire is currently bu...

  14. Crawler-Transporter Time-Lapse

    NASA Video Gallery

    Time-lapse video shows crawler-transporter No. 2 traveling from the Vehicle Assembly Building to Launch Pad 39A at NASA's Kennedy Space Center in Florida. The move was performed by the Ground Syste...

  15. Quantitative 3D structured illumination microscopy of nuclear structures.

    PubMed

    Kraus, Felix; Miron, Ezequiel; Demmerle, Justin; Chitiashvili, Tsotne; Budco, Alexei; Alle, Quentin; Matsuda, Atsushi; Leonhardt, Heinrich; Schermelleh, Lothar; Markaki, Yolanda

    2017-05-01

    3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.

  16. Single molecule microscopy in 3D cell cultures and tissues.

    PubMed

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

  17. Resolution in 3D in multifocal plane microscopy

    NASA Astrophysics Data System (ADS)

    Chao, Jerry; Ram, Sripad; Abraham, Anish V.; Ward, E. Sally; Ober, Raimund J.

    2008-02-01

    Using single molecule microscopy, biological interactions can be imaged and studied at the level of individual biomolecules. When characterizing an imaged biological interaction, the distance separating the two participating biomolecules can provide valuable information. Therefore, the resolvability of an imaging setup is of practical significance in the analysis of the acquired image data. Importantly, the resolvability of the imaging setup needs evaluation in the 3D context, since in general biomolecules reside in 3D space within the cellular environment. We recently introduced an information-theoretic 2D resolution measure which shows that the resolution limit due to Rayleigh's criterion can be overcome. This new result predicts that the resolution of optical microscopes is not limited, but rather can be improved with increased photon counts detected from the single molecules. The 2D result was subsequently extended to the 3D context, and the proposed information-theoretic 3D resolution measure can readily be used to determine the resolvability of a conventional single focal plane imaging setup. Here, we consider the 3D resolution measure for a multifocal plane microscope setup, an imaging system which allows the concurrent imaging of multiple focal planes within a specimen. The technique is useful in applications such as the tracking of subcellular objects in 3D. By comparing their 3D resolution measures, we find a two-plane setup to outperform a comparable conventional single-plane setup in resolvability over a range of axial locations for the single molecule pair. Moreover, we investigate and compare the impact of noise on the resolvability of the two setups.

  18. Unsupervised noise removal algorithms for 3-D confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Roysam, Badrinath; Bhattacharjya, Anoop K.; Srinivas, Chukka; Szarowski, Donald H.; Turner, James N.

    1992-06-01

    Fast algorithms are presented for effective removal of the noise artifact in 3-D confocal fluorescence microscopy images of extended spatial objects such as neurons. The algorithms are unsupervised in the sense that they automatically estimate and adapt to the spatially and temporally varying noise level in the microscopy data. An important feature of the algorithms is the fact that a 3-D segmentation of the field emerges jointly with the intensity estimate. The role of the segmentation is to limit any smoothing to the interiors of regions and hence avoid the blurring that is associated with conventional noise removal algorithms. Fast computation is achieved by parallel computation methods, rather than by algorithmic or modelling compromises. The noise-removal proceeds iteratively, starting from a set of approximate user- supplied, or default initial guesses of the underlying random process parameters. An expectation maximization algorithm is used to obtain a more precise characterization of these parameters, that are then input to a hierarchical estimation algorithm. This algorithm computes a joint solution of the related problems corresponding to intensity estimation, segmentation, and boundary-surface estimation subject to a combination of stochastic priors and syntactic pattern constraints. Three-dimensional stereoscopic renderings of processed 3-D images of murine hippocampal neurons are presented to demonstrate the effectiveness of the method. The processed images exhibit increased contrast and significant smoothing and reduction of the background intensity while avoiding any blurring of the neuronal structures.

  19. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  20. Applied 3D printing for microscopy in health science research

    NASA Astrophysics Data System (ADS)

    Brideau, Craig; Zareinia, Kourosh; Stys, Peter

    2015-03-01

    The rapid prototyping capability offered by 3D printing is considered advantageous for commercial applications. However, the ability to quickly produce precision custom devices is highly beneficial in the research laboratory setting as well. Biological laboratories require the manipulation and analysis of delicate living samples, thus the ability to create custom holders, support equipment, and adapters allow the extension of existing laboratory machines. Applications include camera adapters and stage sample holders for microscopes, surgical guides for tissue preparation, and small precision tools customized to unique specifications. Where high precision is needed, especially the reproduction of fine features, a printer with a high resolution is needed. However, the introduction of cheaper, lower resolution commercial printers have been shown to be more than adequate for less demanding projects. For direct manipulation of delicate samples, biocompatible raw materials are often required, complicating the printing process. This paper will examine some examples of 3D-printed objects for laboratory use, and provide an overview of the requirements for 3D printing for this application. Materials, printing resolution, production, and ease of use will all be reviewed with an eye to producing better printers and techniques for laboratory applications. Specific case studies will highlight applications for 3D-printed devices in live animal imaging for both microscopy and Magnetic Resonance Imaging.

  1. Resolution improvement by 3D particle averaging in localization microscopy

    NASA Astrophysics Data System (ADS)

    Broeken, Jordi; Johnson, Hannah; Lidke, Diane S.; Liu, Sheng; Nieuwenhuizen, Robert P. J.; Stallinga, Sjoerd; Lidke, Keith A.; Rieger, Bernd

    2015-03-01

    Inspired by recent developments in localization microscopy that applied averaging of identical particles in 2D for increasing the resolution even further, we discuss considerations for alignment (registration) methods for particles in general and for 3D in particular. We detail that traditional techniques for particle registration from cryo electron microscopy based on cross-correlation are not suitable, as the underlying image formation process is fundamentally different. We argue that only localizations, i.e. a set of coordinates with associated uncertainties, are recorded and not a continuous intensity distribution. We present a method that owes to this fact and that is inspired by the field of statistical pattern recognition. In particular we suggest to use an adapted version of the Bhattacharyya distance as a merit function for registration. We evaluate the method in simulations and demonstrate it on 3D super-resolution data of Alexa 647 labelled to the Nup133 protein in the nuclear pore complex of Hela cells. From the simulations we find suggestions that for successful registration the localization uncertainty must be smaller than the distance between labeling sites on a particle. These suggestions are supported by theoretical considerations concerning the attainable resolution in localization microscopy and its scaling behavior as a function of labeling density and localization precision.

  2. Phase mask optimization for 3D parallax EDF microscopy

    NASA Astrophysics Data System (ADS)

    Beckers, Ingeborg E.; Gierlack, Michael; Höppel, Robert; Landskron, Jürgen

    2014-03-01

    Extended depth-of-field (EDF) microscopy is a well-investigated and very simple method to obtain projection images with an extended depth of focus. Despite its advantages of being a real-time method applicable to any microscopic mode with high lateral resolution that can be simply realized by extending a commercial microscope, the lack of z-correlation is still a problem. In this work we present a combined technique of EDF and stereomicroscopy. By cross-correlation depth information is obtained. Finally, 3D images are reconstructed for best phase masks and simulation results are evaluated experimentally.

  3. 3D scanning Hall probe microscopy with 700 nm resolution

    NASA Astrophysics Data System (ADS)

    Dede, M.; Akram, R.; Oral, A.

    2016-10-01

    In this report, we present a three dimensional (3D) imaging of magnetic field vector B → (x,y,z) emanating from the magnetic material surfaces using a scanning Hall probe microscopy (3D-SHPM) down to a 700 nm spatial resolution. The Hall probe is used to measure Bz(x,y) on the specimen surface at different heights with the step size of Δz = 250 nm, as we move away from the surface in z direction, until the field decays to zero. These set of images are then used to get ∂Bz(x,y)/∂x and ∂Bz(x,y)/∂y at different z by numerical differentiation. Using the Maxwell's equations in the source free region, Bx(x,y) and By(x,y) can be calculated by integrating ∂Bz(x,y)/∂x and ∂Bz(x,y)/∂y in the z direction. Alternatively, the gradients can also be measured in the Hall gradiometer configuration directly. The operation of the 3D-SHPM is demonstrated by imaging Bx(x,y), By(x,y) and Bz(x,y) on a hard disk specimen at a 700 nm resolution, using both of these methods at 77 K. The system is capable of operating from 300 K down to 4 K range.

  4. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  5. 3D high resolution pure optical photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Xie, Zhixing; Chen, Sung-Liang; Ling, Tao; Guo, L. Jay; Carson, Paul L.; Wang, Xueding

    2012-02-01

    The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After some refinedment of in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM of high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5μm and an axial resolution of 8μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue

  6. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  7. 3D Image Analysis of Geomaterials using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Mulukutla, G.; Proussevitch, A.; Sahagian, D.

    2009-05-01

    Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

  8. 3D geometry-based quantification of colocalizations in multichannel 3D microscopy images of human soft tissue tumors.

    PubMed

    Wörz, Stefan; Sander, Petra; Pfannmöller, Martin; Rieker, Ralf J; Joos, Stefan; Mechtersheimer, Gunhild; Boukamp, Petra; Lichter, Peter; Rohr, Karl

    2010-08-01

    We introduce a new model-based approach for automatic quantification of colocalizations in multichannel 3D microscopy images. The approach uses different 3D parametric intensity models in conjunction with a model fitting scheme to localize and quantify subcellular structures with high accuracy. The central idea is to determine colocalizations between different channels based on the estimated geometry of the subcellular structures as well as to differentiate between different types of colocalizations. A statistical analysis was performed to assess the significance of the determined colocalizations. This approach was used to successfully analyze about 500 three-channel 3D microscopy images of human soft tissue tumors and controls.

  9. Holographic microscopy for 3D tracking of bacteria

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Cho, Yong Bin; El-Kholy, Marwan; Bedrossian, Manuel; Rider, Stephanie; Lindensmith, Christian; Wallace, J. Kent

    2016-03-01

    Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

  10. New Siemens Research Turbine - time lapse

    SciTech Connect

    2009-01-01

    The National Renewable Energy Laboratory (NREL) and Siemens Energy Inc. recently commissioned a new 2.3 megawatt Siemens wind turbine at NREL's National Wind Technology Center. This video shows a time lapse of the installation. The turbine is the centerpiece of a multi-year project to study the performance and aerodynamics of a new class of large, land-based machines — in what will be the biggest government-industry research partnership for wind power generation ever undertaken in the U.S.

  11. 3D super-resolution imaging by localization microscopy.

    PubMed

    Magenau, Astrid; Gaus, Katharina

    2015-01-01

    Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

  12. Precision 3-D microscopy with intensity modulated fibre optic scanners

    NASA Astrophysics Data System (ADS)

    Olmos, P.

    2016-01-01

    Optical 3-D imagers constitute a family of precision and useful instruments, easily available on the market in a wide variety of configurations and performances. However, besides their cost they usually provide an image of the object (i.e. a more or less faithful representation of the reality) instead of a truly object's reconstruction. Depending on the detailed working principles of the equipment, this reconstruction may become a challenging task. Here a very simple yet reliable device is described; it is able to form images of opaque objects by illuminating them with an optical fibre and collecting the reflected light with another fibre. Its 3-D capability comes from the spatial filtering imposed by the fibres together with their movement (scanning) along the three directions: transversal (surface) and vertical. This unsophisticated approach allows one to model accurately the entire optical process and to perform the desired reconstruction, finding that information about the surface which is of interest: its profile and its reflectance, ultimately related to the type of material.

  13. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

    PubMed Central

    Spiegelhalter, Coralie; Tosch, Valérie; Hentsch, Didier; Koch, Marc; Kessler, Pascal; Schwab, Yannick; Laporte, Jocelyn

    2010-01-01

    Background In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM. PMID:20140253

  14. Non-destructive mapping of grain orientations in 3D by laboratory X-ray microscopy

    PubMed Central

    McDonald, S. A.; Reischig, P.; Holzner, C.; Lauridsen, E. M.; Withers, P. J.; Merkle, A. P.; Feser, M.

    2015-01-01

    The ability to characterise crystallographic microstructure, non-destructively and in three-dimensions, is a powerful tool for understanding many aspects related to damage and deformation mechanisms in polycrystalline materials. To this end, the technique of X-ray diffraction contrast tomography (DCT) using monochromatic synchrotron and polychromatic laboratory X-ray sources has been shown to be capable of mapping crystal grains and their orientations non-destructively in 3D. Here we describe a novel laboratory-based X-ray DCT modality (LabDCT), enabling the wider accessibility of the DCT technique for routine use and in-depth studies of, for example, temporal changes in crystallographic grain structure non-destructively over time through ‘4D’ in situ time-lapse studies. The capability of the technique is demonstrated by studying a titanium alloy (Ti-β21S) sample. In the current implementation the smallest grains that can be reliably detected are around 40 μm. The individual grain locations and orientations are reconstructed using the LabDCT method and the results are validated against independent measurements from phase contrast tomography and electron backscatter diffraction respectively. Application of the technique promises to provide important insights related to the roles of recrystallization and grain growth on materials properties as well as supporting 3D polycrystalline modelling of materials performance. PMID:26494523

  15. Saturn Time Lapse - Target of Opportunity

    NASA Astrophysics Data System (ADS)

    Westphal, J.

    1990-12-01

    This program will obtain sets of exposures of Saturn in several spectral bands over a time span of about a week. By selecting filters that are sensitive to atmospheric processes (methane absorption and molecular scattering), the dynamic behavior can be mapped at several altitudes within the Saturnian atmosphere. This sequence will utilize time lapse sequences in chip PC-6 to continue monitoring motions associated with the great white spot that was discovered in late September. These observations will allow us to follow the evolution of the storm. Observation is broken up into 2 parts - part 1 = Get as many orbits as possible in a 31 orbit timeframe. Part 2 = Get as many orbits as possible in a 16 orbit timeframe. Use the FGS SAA avoidance contour versus the WF/PC SAA avoidance contour. During orbits with FGS SAA impact, obtain earth flats, using seq FLATn. Take flats in each filter (FLAT1->FLAT6) then go back and take second set in each filter, time permitting.

  16. Cytology 3D structure formation based on optical microscopy images

    NASA Astrophysics Data System (ADS)

    Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

  17. Registration and 3D visualization of large microscopy images

    NASA Astrophysics Data System (ADS)

    Mosaliganti, Kishore; Pan, Tony; Sharp, Richard; Ridgway, Randall; Iyengar, Srivathsan; Gulacy, Alexandra; Wenzel, Pamela; de Bruin, Alain; Machiraju, Raghu; Huang, Kun; Leone, Gustavo; Saltz, Joel

    2006-03-01

    Inactivation of the retinoblastoma gene in mouse embryos causes tissue infiltrations into critical sections of the placenta, which has been shown to affect fetal survivability. Our collaborators in cancer genetics are extremely interested in examining the three dimensional nature of these infiltrations given a stack of two dimensional light microscopy images. Three sets of wildtype and mutant placentas was sectioned serially and digitized using a commercial light microscopy scanner. Each individual placenta dataset consisted of approximately 1000 images totaling 700 GB in size, which were registered into a volumetric dataset using National Library of Medicine's (NIH/NLM) Insight Segmentation and Registration Toolkit (ITK). This paper describes our method for image registration to aid in volume visualization of tissue level intermixing for both wildtype and Rb - specimens. The registration process faces many challenges arising from the large image sizes, damages during sectioning, staining gradients both within and across sections, and background noise. These issues limit the direct application of standard registration techniques due to frequent convergence to local solutions. In this work, we develop a mixture of automated and semi-automated enhancements with ground-truth validation for the mutual information-based registration algorithm. Our final volume renderings clearly show tissue intermixing differences between both wildtype and Rb - specimens which are not obvious prior to registration.

  18. Two-Layer Elastographic 3-D Traction Force Microscopy

    NASA Astrophysics Data System (ADS)

    Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A.; Lasheras, Juan C.; Del Álamo, Juan C.

    2017-01-01

    Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

  19. Two-Layer Elastographic 3-D Traction Force Microscopy

    PubMed Central

    Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A.; Lasheras, Juan C.; del Álamo, Juan C.

    2017-01-01

    Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions. PMID:28074837

  20. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

    PubMed Central

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-01-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm. PMID:27375935

  1. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

  2. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    PubMed

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  3. Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

    PubMed Central

    Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge

    2015-01-01

    Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color. PMID:26153696

  4. Observation of infiltration experiments with time lapse electrical resistivity tomography

    NASA Astrophysics Data System (ADS)

    Noell, Ursula; Ganz, Christina; Altfelder, Sven; Günther, Thomas; Duijnisveld, Wilhelmus; Grissemann, Christoph

    2010-05-01

    Recent progress in the development of resistivity equipment enables the real time observation of infiltration processes through the vadose zone. In order to study the advantages and limitations of the method infiltration experiments are carried out for different soil types at various locations. All sites are subsequently excavated and investigated in detail. For an improved verification of the resistivity data the most recent experiment is conducted using a colour tracer. Two infiltration experiments are carried out in sandy soil. The location is Fuhrberg, close to Hannover, Germany. The area has been intensively studied for soil research purposes for more than 30 years. During both infiltration experiments water (110 l/80 l) is infiltrated for a period of 4.5 h and 8 h, respectively, and the infiltration process is observed by ERT. The resistivity measurements are conducted using a 3D-dipole-dipole configuration with electrode distances of 20 cm in the centre of the infiltration field. The whole resistivity array consists of 200 and 300 electrodes, respectively. The second experiment uses increased electrode spacing in the border area in order to enable the resolution of the deeper groundwater table (3.5 m during the second experiment compared to about 1.2 m for the first experiment). Immediately after completion of the resistivity measurements TDR and tensiometer measurements are carried out in 5-8 slices of the excavated infiltration area over a period of several days. The colour tracer used during the second experiment clearly outlines the infiltration plume with sharp outer limits. The ERT inversion depicts the shape of the plume successfully. Time lapse ERT interpretation reveals the development of the plume in time. The combination of ERT interpretation and TDR measurements enables the construction of the relationship between water content and resistivity as reconstructed by ERT using an Archie approach. By using this function water content changes can be

  5. Atlantis Time-Lapse Move to KSC Visitor Complex

    NASA Video Gallery

    Time-lapse cameras captured space shuttle Atlantis making a 10-mile trek from the Vehicle Assembly Building at NASA's Kennedy Space Center in Florida to the Kennedy Space Center Visitor Complex whe...

  6. Time-Lapsed Animation of a Mercury Day

    NASA Video Gallery

    Parts of Prokofiev crater (center) and Kandinsky crater (upper left side of Prokofiev) stay in darkness, making it possible for ice to persist on the surface. This time-lapsed animation represents ...

  7. NEA Scout Solar Sail: Half-scale Fold Time Lapse

    NASA Video Gallery

    In this time lapse, the Near-Earth Asteroid Scout (NEA Scout) CubeSat team rolls a half-scale prototype of the small satellite's solar sail in preparation for a deployment test. During its mission,...

  8. Confocal time lapse imaging as an efficient method for the cytocompatibility evaluation of dental composites.

    PubMed

    Attik, Ghania Nina; Gritsch, Kerstin; Colon, Pierre; Grosgogeat, Brigitte

    2014-11-09

    It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

  9. Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

    PubMed

    Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

    2013-07-01

    Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

  10. Volumetric monitoring of aqueous two phase system droplets using time-lapse optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Lee, J.; Bathany, C.; Ahn, Y.; Takayama, S.; Jung, W.

    2016-02-01

    We present a volumetric monitoring method to observe the morphological changes of aqueous two phase system (ATPS) droplets in a microfluidic system. Our method is based on time-lapse optical coherence tomography (OCT) which allows the study of the dynamics of ATPS droplets while visualizing their 3D structures and providing quantitative information on the droplets. In this study, we monitored the process of rehydration and deformation of an ATPS droplet in a microfluidic system and quantified the changes of its volume and velocity under both static and dynamic fluid conditions. Our results indicate that time-lapse OCT is a very promising tool to evaluate the unprecedented features of droplet-based microfluidics.

  11. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    PubMed

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

  12. Optimum conditions for high-quality 3D reconstruction in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Taehoon; Kim, Taejoong; Lee, SeungWoo; Gweon, Dae-Gab; Seo, Jungwoo

    2006-02-01

    Confocal Scanning Microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning Microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable n-th order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

  13. Quantitative IR microscopy and spectromics open the way to 3D digital pathology.

    PubMed

    Bobroff, Vladimir; Chen, Hsiang-Hsin; Delugin, Maylis; Javerzat, Sophie; Petibois, Cyril

    2016-06-01

    Currently, only mass-spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro-microscopy is reported. An automated curve-fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR-absorption spectrum into a IR-band spectrum; (3) the reconstruction of an 3D IR-band matrix (x, y, z for voxel position and a 4(th) dimension with all IR-band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

  14. Novel scanning electron microscopy methods for analyzing the 3D structure of the Golgi apparatus.

    PubMed

    Koga, Daisuke; Ushiki, Tatsuo; Watanabe, Tsuyoshi

    2017-01-01

    The structure of the Golgi apparatus has been extensively examined by light and electron microscopy, but details of its three-dimensional (3D) structure have remained unclear because of the technical limitations of conventional microscopy techniques. To overcome this problem, we have developed several novel scanning electron microscopy (SEM) methods for observing the 3D structure of subcellular organelles including the Golgi apparatus: (1) an osmium maceration method that facilitates SEM observation of membranous organelles, including the Golgi apparatus, by selectively removing soluble cytoplasmic proteins, (2) an osmium impregnation/maceration method that combines an osmium impregnation method with the osmium maceration method to determine the polarity of the Golgi apparatus by SEM, (3) a correlative light and SEM method that combines a cryosectioning technique with the osmium maceration method to enable correlation of the immunocytochemical distribution of molecules with the 3D ultrastructure of the Golgi apparatus, and (4) array tomography based on the systematic collection and integration of SEM images of serial ultrathin sections on glass slides for revealing the 3D ultrastructure of the entire Golgi apparatus. Together, the novel SEM techniques listed above can reveal the complete 3D structure of the Golgi apparatus in different cell types.

  15. 3D image reconstruction algorithms for cryo-electron-microscopy images of virus particles

    NASA Astrophysics Data System (ADS)

    Doerschuk, Peter C.; Johnson, John E.

    2000-11-01

    A statistical model for the object and the complete image formation process in cryo electron microscopy of viruses is presented. Using this model, maximum likelihood reconstructions of the 3D structure of viruses are computed using the expectation maximization algorithm and an example based on Cowpea mosaic virus is provided.

  16. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

    PubMed Central

    Harney, Allison S.; Wang, Yarong; Condeelis, John S.; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  17. TIME-LAPSE SEISMIC MODELING & INVERSION OF CO2 SATURATION FOR SEQUESTRATION AND ENHANCED OIL RECOVERY

    SciTech Connect

    Mark A. Meadows

    2006-03-31

    Injection of carbon dioxide (CO2) into subsurface aquifers for geologic storage/sequestration, and into subsurface hydrocarbon reservoirs for enhanced oil recovery, has become an important topic to the nation because of growing concerns related to global warming and energy security. In this project we developed new ways to predict and quantify the effects of CO2 on seismic data recorded over porous reservoir/aquifer rock systems. This effort involved the research and development of new technology to: (1) Quantitatively model the rock physics effects of CO2 injection in porous saline and oil/brine reservoirs (both miscible and immiscible). (2) Quantitatively model the seismic response to CO2 injection (both miscible and immiscible) from well logs (1D). (3) Perform quantitative inversions of time-lapse 4D seismic data to estimate injected CO2 distributions within subsurface reservoirs and aquifers. This work has resulted in an improved ability to remotely monitor the injected CO2 for safe storage and enhanced hydrocarbon recovery, predict the effects of CO2 on time-lapse seismic data, and estimate injected CO2 saturation distributions in subsurface aquifers/reservoirs. We applied our inversion methodology to a 3D time-lapse seismic dataset from the Sleipner CO2 sequestration project, Norwegian North Sea. We measured changes in the seismic amplitude and traveltime at the top of the Sleipner sandstone reservoir and used these time-lapse seismic attributes in the inversion. Maps of CO2 thickness and its standard deviation were generated for the topmost layer. From this information, we estimated that 7.4% of the total CO2 injected over a five-year period had reached the top of the reservoir. This inversion approach could also be applied to the remaining levels within the anomalous zone to obtain an estimate of the total CO2 injected.

  18. Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

    PubMed Central

    Boudejltia, Karim Zouaoui; Ribeiro de Sousa, Daniel; Uzureau, Pierrick; Yourassowsky, Catherine; Perez-Morga, David; Courbebaisse, Guy; Chopard, Bastien; Dubois, Frank

    2015-01-01

    Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

  19. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    PubMed Central

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

  20. Deep Learning Segmentation of Optical Microscopy Images Improves 3D Neuron Reconstruction.

    PubMed

    Li, Rongjian; Zeng, Tao; Peng, Hanchuan; Ji, Shuiwang

    2017-03-08

    Digital reconstruction, or tracing, of 3-dimensional (3D) neuron structure from microscopy images is a critical step toward reversing engineering the wiring and anatomy of a brain. Despite a number of prior attempts, this task remains very challenging, especially when images are contaminated by noises or have discontinued segments of neurite patterns. An approach for addressing such problems is to identify the locations of neuronal voxels using image segmentation methods prior to applying tracing or reconstruction techniques. This preprocessing step is expected to remove noises in the data, thereby leading to improved reconstruction results. In this work, we proposed to use 3D Convolutional neural networks (CNNs) for segmenting the neuronal microscopy images. Specifically, we designed a novel CNN architecture that takes volumetric images as the inputs and their voxel-wise segmentation maps as the outputs. The developed architecture allows us to train and predict using large microscopy images in an end-to-end manner. We evaluated the performance of our model on a variety of challenging 3D microscopy images from different organisms. Results showed that the proposed methods improved the tracing performance significantly when combined with different reconstruction algorithms.

  1. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  2. Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Netti, Paolo A.; Coppola, Giuseppe; Ferraro, Pietro

    2013-04-01

    We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

  3. Staining and embedding of human chromosomes for 3-d serial block-face scanning electron microscopy.

    PubMed

    Yusuf, Mohammed; Chen, Bo; Hashimoto, Teruo; Estandarte, Ana Katrina; Thompson, George; Robinson, Ian

    2014-12-01

    The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic chromosomes using mostly 2-D microscopy methods. To image micron-sized human chromosomes in 3-D, we developed a procedure for preparing samples for serial block-face scanning electron microscopy (SBFSEM). Polyamine chromosomes are first separated using a simple filtration method and then stained with heavy metal. We show that the DNA-specific platinum blue provides higher contrast than osmium tetroxide. A two-step procedure for embedding chromosomes in resin is then used to concentrate the chromosome samples. After stacking the SBFSEM images, a familiar X-shaped chromosome was observed in 3-D.

  4. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

    PubMed

    Roselli, L; Paparella, F; Stanca, E; Basset, A

    2015-06-01

    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  5. Infrared differential interference contrast microscopy for 3D interconnect overlay metrology.

    PubMed

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-08-12

    One of the main challenges for 3D interconnect metrology of bonded wafers is measuring through opaque silicon wafers using conventional optical microscopy. We demonstrate here the use infrared microscopy, enhanced by implementing the differential interference contrast (DIC) technique, to measure the wafer bonding overlay. A pair of two dimensional symmetric overlay marks were processed at both the front and back sides of thinned wafers to evaluate the bonding overlay. A self-developed analysis algorithm and theoretical fitting model was used to map the overlay error between the bonded wafers and the interconnect structures. The measurement accuracy was found to be better than 1.0 micron.

  6. Photometry unlocks 3D information from 2D localization microscopy data.

    PubMed

    Franke, Christian; Sauer, Markus; van de Linde, Sebastian

    2017-01-01

    We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

  7. Using 3D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

    DTIC Science & Technology

    2014-05-01

    microenvironments on breast cancer by creating arrays of polydimethlysiloxane (PDMS) microposts of different stiffness and sizes and seeded them with MCF-7 cells...of MCF-7s. Finally, with QPI, we investigated the real-time response of breast- cancer cells to different microenvironmental cues . We thus have...controls this cellular phenotype. To realize this goal, we had proposed to use 3D super-resolution microscopy to visualize how individual breast CaSCs

  8. Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

    PubMed

    Schmid, Volker J; Cremer, Marion; Cremer, Thomas

    2017-03-18

    Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.

  9. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide.

    PubMed

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-10

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  10. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    PubMed Central

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-01-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

  11. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    NASA Astrophysics Data System (ADS)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  12. Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

    2015-03-01

    Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

  13. Monitoring hydraulic processes with automated time-lapse electrical resistivity tomography (ALERT)

    NASA Astrophysics Data System (ADS)

    Kuras, Olivier; Pritchard, Jonathan D.; Meldrum, Philip I.; Chambers, Jonathan E.; Wilkinson, Paul B.; Ogilvy, Richard D.; Wealthall, Gary P.

    2009-10-01

    Hydraulic processes in porous media can be monitored in a minimally invasive fashion by time-lapse electrical resistivity tomography (ERT). The permanent installation of specifically designed ERT instrumentation, telemetry and information technology (IT) infrastructure enables automation of data collection, transfer, processing, management and interpretation. Such an approach gives rise to a dramatic increase in temporal resolution, thus providing new insight into rapidly occurring subsurface processes. In this paper, we discuss a practical implementation of automated time-lapse ERT. We present the results of a recent study in which we used controlled hydraulic experiments in two test cells at reduced field scale to explore the limiting conditions for process monitoring with cross-borehole ERT measurements. The first experiment used three adjacent boreholes to monitor rapidly rising and falling water levels. For the second experiment, we injected a saline tracer into a homogeneous flow field in freshwater-saturated sand; the dynamics of the plume were then monitored with 2D measurements across a 9-borehole fence and 3D measurements across a 3 × 3 grid of boreholes. We investigated different strategies for practical data acquisition and show that simple re-ordering of ERT measurement schemes can help harmonise data collection with the nature of the monitored process. The methodology of automated time-lapse ERT was found to perform well in different monitoring scenarios (2D/3D plus time) at time scales associated with realistic subsurface processes. The limiting factor is the finite amount of time needed for the acquisition of sufficiently comprehensive datasets. We found that, given the complexity of our monitoring scenarios, typical frame rates of at least 1.5-3 images per hour were possible without compromising image quality.

  14. Epikarstic storage and doline structural characterization with time-lapse geophysics (seismic refraction & electrical resistivity)

    NASA Astrophysics Data System (ADS)

    Valois, R.; Galibert, P.; Guérin, R.; Mendes, M.; Plagnes, V.

    2011-12-01

    Karst formations are one of the most challenging environments in terms of groundwater, engineering and environmental issues. Geophysical methods can provide useful subsurface information in karst regions concerning groundwater vulnerability assessment, exploitation or hazard estimation. First, dolines are studied as preferential pathways for the protection of karstic aquifer in south France. Geophysics helps to characterize lateral and underground morphologies of such objects and is able to detect doline hidden by the soil cover too. Electrical resistivity and seismic refraction tomographies provide information about dolines filling and could help to propose a genesis scenario. Time-lapse resistivity measurements show that the studied doline is more vulnerable to infiltration on its sides than at its centre. The epikarst could be defined as a perched aquifer above the massive carbonate rocks; it constitutes a highly fractured zone, which water stock capacities. So, the epikarst was investigated with 3D seismic refraction and results show an important velocity anisotropy linked to the fracturing and weathering of the dolostone. The 3D model presents also some large heterogeneities: a corridor with highly weathered dolostone and an unweathered pinnacle. The corridor is probably situated on vertical joints, which have conducted aggressive water. The associated weathering with residual weathered-rock keeping its initial volume could create a "ghost-rock" corridor. So, the epikarst in the dolostones of the Causse du Larzac (France) seems to be composed by "ghost-rock" developed around a specific direction of fractures. Time-lapse electrical resistivity and seismic refraction velocity were carried out on this epikarst to observe the influence of water saturation on the measurements. The results show important variations for both seismic and electrical methods and are localized in the first 6 m: in the weathered zone. So, time-lapse measurements could more easily identify

  15. Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids

    PubMed Central

    Rane, Tushar D.; Armani, Andrea M.

    2016-01-01

    Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy. PMID:27936027

  16. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    NASA Astrophysics Data System (ADS)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  17. Point scanning confocal microscopy facilitates 3D human hair follicle imaging in tissue sections.

    PubMed

    Kloepper, Jennifer E; Bíró, Tamás; Paus, Ralf; Cseresnyés, Zoltán

    2010-07-01

    Efficiency is a key factor in determining whether a scientific method becomes widely accepted in practical applications. In dermatology, morphological characterisation of intact hair follicles by traditional methods can be rather inefficient. Samples are embedded, sliced, imaged and digitally reconstructed, which can be time-consuming. Confocal microscopy, on the other hand, is more efficient and readily applicable to study intact hair follicles. Modern confocal microscopes deliver and collect light very efficiently and thus allow high spatial resolution imaging of relatively thick samples. In this letter, we report that we successfully imaged entire intact human hair follicles using point scanning confocal microscopy. Light delivery and light-collection were further improved by preparing the samples in 2,2'-Thiodiethanol (TDE), thus reducing refractive index gradients. The relatively short total scan times and the high quality of the acquired 3D images make confocal microscopy a desirable method for studying intact hair follicles under normal and pathological conditions.

  18. 3D reconstruction of cortical microtubules using multi-angle total internal reflection fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Luhong; Xiu, Peng; Zhou, Xiaoxu; Fan, Jiannan; Kuang, Cuifang; Liu, Xu; Xu, Yingke

    2017-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different illumination angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve 40 nm axial resolution in experimental conditions where SNR is as low as 2, with 35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.

  19. Time-lapse Raman imaging of osteoblast differentiation

    NASA Astrophysics Data System (ADS)

    Hashimoto, Aya; Yamaguchi, Yoshinori; Chiu, Liang-Da; Morimoto, Chiaki; Fujita, Katsumasa; Takedachi, Masahide; Kawata, Satoshi; Murakami, Shinya; Tamiya, Eiichi

    2015-07-01

    Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that β-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.

  20. Imaging the behavior of molecules in biological systems: breaking the 3D speed barrier with 3D multi-resolution microscopy.

    PubMed

    Welsher, Kevin; Yang, Haw

    2015-01-01

    The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.

  1. Computational methods for constructing protein structure models from 3D electron microscopy maps.

    PubMed

    Esquivel-Rodríguez, Juan; Kihara, Daisuke

    2013-10-01

    Protein structure determination by cryo-electron microscopy (EM) has made significant progress in the past decades. Resolutions of EM maps have been improving as evidenced by recently reported structures that are solved at high resolutions close to 3Å. Computational methods play a key role in interpreting EM data. Among many computational procedures applied to an EM map to obtain protein structure information, in this article we focus on reviewing computational methods that model protein three-dimensional (3D) structures from a 3D EM density map that is constructed from two-dimensional (2D) maps. The computational methods we discuss range from de novo methods, which identify structural elements in an EM map, to structure fitting methods, where known high resolution structures are fit into a low-resolution EM map. A list of available computational tools is also provided.

  2. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    PubMed

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

  3. 3D structure tensor analysis of light microscopy data for validating diffusion MRI

    PubMed Central

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A.; Kohama, Steven G.; Jespersen, Sune Nørhøj; Kroenke, Christopher D.

    2015-01-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image “stacks” acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations

  4. Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Grover, Ginni

    In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are

  5. Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation.

    PubMed

    Burkovskiy, I; Lehmann, C; Jiang, C; Zhou, J

    2016-11-01

    Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single-step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.

  6. Applications of Digital Holography: From Microscopy to 3D-Television

    NASA Astrophysics Data System (ADS)

    Kreis, T.

    2012-03-01

    The paper gives an overview of the applications of digital holography based on the one hand on CCD-recording, computer storage, and numerical reconstruction of the wave fields, and on the other hand on numerical calculation of computer generated holograms (CGH) and the transfer of these CGHs to spatial light modulators (SLM) for optical reconstruction of the wave fields. The first mentioned type of digital holography finds applications in digital holographic microscopy, particle analysis, and interferometric form and deformation measurement, while the second type constitutes the basis for holographic 3D TV. The space-bandwidth-problem occuring in this context is addressed and first partial solutions are presented.

  7. X-ray Laue Diffraction Microscopy in 3D at the Advanced Photon Source

    SciTech Connect

    Liu, W.; Zschack, P.; Tischler, Jonathan Zachary; Ice, Gene E; Larson, Ben C

    2011-01-01

    Studies of materials on mesoscopic length-scales require a penetrating structural probe with submicron point-to-point spatial resolution. The principle research activities at beamline 34-ID-E of the Advanced Photon Source (APS) involve development of exciting new micro-/nano-diffraction techniques for characterization and microscopy in support of both applied engineering and fundamental materials research. Taking advantage of the high brightness of the source, advanced focusing mirrors, a novel depth profiling technique, and high-speed area detectors, three-dimensional scanning Laue diffraction microscopy provides detailed local structural information of crystalline materials, such as crystallographic orientation, orientation gradients, and strain tensors. It is general and applicable to single-crystal, polycrystalline, composite, deformed, and functionally graded materials. Applications include 3D diffraction investigations for a diverse and growing user community with interests in materials deformation, electro-migration, recrystallization, fatigue, solid-solution precipitation, high-pressure environments, and condensed matter physics.

  8. Cellulose Nanocrystals as Chiral Inducers: Enantioselective Catalysis and Transmission Electron Microscopy 3D Characterization.

    PubMed

    Kaushik, Madhu; Basu, Kaustuv; Benoit, Charles; Cirtiu, Ciprian M; Vali, Hojatollah; Moores, Audrey

    2015-05-20

    Cellulose nanocrystals (CNCs), derived from cellulose, provide us with an opportunity to devise more sustainable solutions to current technological challenges. Enantioselective catalysis, especially heterogeneous, is the preferred method for the synthesis of pure chiral molecules in the fine chemical industries. Cellulose has been long sought as a chiral inducer in enantioselective catalysis. We report herein an unprecedentedly high enantiomeric excess (ee) for Pd patches deposited onto CNCs used as catalysts for the hydrogenation of prochiral ketones in water at room temperature and 4 bar H2. Our system, where CNCs acted as support and sole chiral source, achieved an ee of 65% with 100% conversions. Cryo-electron microscopy, high-resolution transmission electron microscopy, and tomography were used for the first time to study the 3D structure of a metal functionalized CNC hybrid. It established the presence of sub-nanometer-thick Pd patches at the surface of CNCs and provided insight into the chiral induction mechanism.

  9. Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

    PubMed

    Chen, Xueqin; Grégoire, Sébastien; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-02-28

    Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products.

  10. 3D Imaging of Diatoms with Ion-abrasion Scanning Electron Microscopy

    PubMed Central

    Hildebrand, Mark; Kim, Sang; Shi, Dan; Scott, Keana; Subramaniam, Sriram

    2009-01-01

    Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at ~ 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system. PMID:19269330

  11. X-ray microscopy for in situ characterization of 3D nanostructural evolution in the laboratory

    NASA Astrophysics Data System (ADS)

    Hornberger, Benjamin; Bale, Hrishikesh; Merkle, Arno; Feser, Michael; Harris, William; Etchin, Sergey; Leibowitz, Marty; Qiu, Wei; Tkachuk, Andrei; Gu, Allen; Bradley, Robert S.; Lu, Xuekun; Withers, Philip J.; Clarke, Amy; Henderson, Kevin; Cordes, Nikolaus; Patterson, Brian M.

    2015-09-01

    X-ray microscopy (XRM) has emerged as a powerful technique that reveals 3D images and quantitative information of interior structures. XRM executed both in the laboratory and at the synchrotron have demonstrated critical analysis and materials characterization on meso-, micro-, and nanoscales, with spatial resolution down to 50 nm in laboratory systems. The non-destructive nature of X-rays has made the technique widely appealing, with potential for "4D" characterization, delivering 3D micro- and nanostructural information on the same sample as a function of sequential processing or experimental conditions. Understanding volumetric and nanostructural changes, such as solid deformation, pore evolution, and crack propagation are fundamental to understanding how materials form, deform, and perform. We will present recent instrumentation developments in laboratory based XRM including a novel in situ nanomechanical testing stage. These developments bridge the gap between existing in situ stages for micro scale XRM, and SEM/TEM techniques that offer nanometer resolution but are limited to analysis of surfaces or extremely thin samples whose behavior is strongly influenced by surface effects. Several applications will be presented including 3D-characterization and in situ mechanical testing of polymers, metal alloys, composites and biomaterials. They span multiple length scales from the micro- to the nanoscale and different mechanical testing modes such as compression, indentation and tension.

  12. Rapid reconstruction of 3D neuronal morphology from light microscopy images with augmented rayburst sampling.

    PubMed

    Ming, Xing; Li, Anan; Wu, Jingpeng; Yan, Cheng; Ding, Wenxiang; Gong, Hui; Zeng, Shaoqun; Liu, Qian

    2013-01-01

    Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.

  13. 3D X-ray ultra-microscopy of bone tissue.

    PubMed

    Langer, M; Peyrin, F

    2016-02-01

    We review the current X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. We further review the different ultra-structural features that have so far been resolved: the lacuno-canalicular network, collagen orientation, nano-scale mineralization and their use as basis for mechanical simulations. X-ray computed tomography at the micro-metric scale is increasingly considered as the reference technique in imaging of bone micro-structure. The trend has been to push towards increasingly higher resolution. Due to the difficulty of realizing optics in the hard X-ray regime, the magnification has mainly been due to the use of visible light optics and indirect detection of the X-rays, which limits the attainable resolution with respect to the wavelength of the visible light used in detection. Recent developments in X-ray optics and instrumentation have allowed to implement several types of methods that achieve imaging that is limited in resolution by the X-ray wavelength, thus enabling computed tomography at the nano-scale. We review here the X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. Further, we review the different ultra-structural features that have so far been resolved and the applications that have been reported: imaging of the lacuno-canalicular network, direct analysis of collagen orientation, analysis of mineralization on the nano-scale and use of 3D images at the nano-scale to drive mechanical simulations. Finally, we discuss the issue of going beyond qualitative description to quantification of ultra-structural features.

  14. Comparison of 3D Orientation Distribution Functions Measured with Confocal Microscopy and Diffusion MRI

    PubMed Central

    Schilling, Kurt; Janve, Vaibhav; Gao, Yurui; Stepniewska, Iwona; Landman, Bennett A; Anderson, Adam W

    2016-01-01

    The ability of diffusion MRI (dMRI) fiber tractography to non-invasively map three-dimensional (3D) anatomical networks in the human brain has made it a valuable tool in both clinical and research settings. However, there are many assumptions inherent to any tractography algorithm that can limit the accuracy of the reconstructed fiber tracts. Among them is the assumption that the diffusion-weighted images accurately reflect the underlying fiber orientation distribution (FOD) in the MRI voxel. Consequently, validating dMRI’s ability to assess the underlying fiber orientation in each voxel is critical for its use as a biomedical tool. Here, using post-mortem histology and confocal microscopy, we present a method to perform histological validation of orientation functions in 3D, which has previously been limited to two-dimensional analysis of tissue sections. We demonstrate the ability to extract the 3D FOD from confocal z-stacks, and quantify the agreement between the MRI estimates of orientation information obtained using constrained spherical deconvolution (CSD) and the true geometry of the fibers. We find an orientation error of approximately 6° in voxels containing nearly parallel fibers, and 10-11° in crossing fiber regions, and note that CSD was unable to resolve fibers crossing at angles below 60° in our dataset. This is the first time the 3D white matter orientation distribution is calculated from histology and compared to dMRI. Thus, this technique serves as a gold standard for dMRI validation studies - providing the ability to determine the extent to which the dMRI signal is consistent with the histological FOD, and to establish how well different dMRI models can predict the ground truth FOD. PMID:26804781

  15. Improved site contamination through time-lapse complex resistivity imaging

    NASA Astrophysics Data System (ADS)

    Flores Orozco, Adrian; Kemna, Andreas; Cassiani, Giorgio; Binley, Andrew

    2016-04-01

    In the framework of the EU FP7 project ModelPROBE, time-lapse complex resistivity (CR) measurements were conducted at a test site close to Trecate (NW Italy). The objective was to investigate the capabilities of the CR imaging method to delineate the geometry and dynamics of subsurface hydrocarbon contaminant plume which resulted from a crude oil spill in 1994. To achieve this it is required to discriminate the electrical signal associated to static (i.e., lithology) from dynamic changes in the subsurface, with the latter associated to significant seasonal groundwater fluctuations. Previous studies have demonstrated the benefits of the CR method to gain information which is not accessible with common electrical resistivity tomography. However field applications are still rarely and neither the analysis of the data error for CR time-lapse measurements, nor the inversion itself haven not received enough attention. While the ultimate objective at the site is to characterize, here we address the discrimination of the lithological and hydrological controls on the IP response by considering data collected in an uncontaminated area of the site. In this study we demonstrate that an adequate error description of CR measurements provides images free of artifacts and quantitative superior than previous approaches. Based on this approach, differential images computed for time-lapse data exhibited anomalies well correlated with spatiotemporal changes correlated to seasonal fluctuations in the groundwater level. The proposed analysis may be useful in the characterization of fate and transport of hydrocarbon contaminants relevant for the site, which presents areas contaminated with crude oil.

  16. CO2 storage in Otway, Australia: influence of geological and reservoir parameters on time-lapse seismic signal modeling

    NASA Astrophysics Data System (ADS)

    Gendrin, A.; Bouquet, S.; Urosevic, M.; Bernth, H.; Kostov, C.; Wisman, P.; Labregere, D.; Verliac, M.

    2009-04-01

    Until now, time lapse seismic signal is mostly used qualitatively in CO2 storage projects, to map the extent of the CO2 plume. Ultimately, we want to use it quantitatively to calculate the amount of injected CO2. This will allow us to check that all the CO2 injected is properly stored and that no leakage is occurring. CO2 injection started in Otway, Australia, in March 2008. Over two years, 100,000 tons of CO2 should be injected in this depleted methane reservoir. This project is very challenging since the injected CO2 mixes with residual methane, which generates a weak time-lapse seismic signature. However, a wealth of information is available: logs, a static model, a calibrated reservoir model, and a rock physics model based on core measurements, which are fundamental for the project. We use 3D finite difference modeling to simulate the expected time-lapse seismic signal. Our initial velocity model is built with logs and seismic interpretation. The rock physics approach is used on the outputs of the reservoir simulation to generate a velocity model after injection. Seismic simulation is then run before and after injection, which provides the theoretical time-lapse seismic signal. Several parameters are poorly constrained in the workflow, which affect time-lapse signal prediction. Porosity and permeability grids are constrained at the wells and are statistically distributed throughout the volume. Relative permeability, capillary pressure, hysteresis, have been measured, for CO2, on a few samples only throughout the world. In this work, we explore the influence of uncertainty on these parameters, and derive an error bar on the estimate of the amount of CO2 stored.

  17. A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

    PubMed Central

    Smit, Theodoor H.

    2016-01-01

    Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture. The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy). PMID:28060338

  18. Using a time lapse microgravity model for mapping seawater intrusion around Semarang

    NASA Astrophysics Data System (ADS)

    Supriyadi, Khumaedi, Yusuf, M.; Agung, W.

    2016-03-01

    A modeling of time-lapse microgravity anomaly due to sea water intrusion has been conducted. It used field data of aquifer cross section, aquifer thickness and lithology of research area. Those data were then processed using Grav3D and Surfer. Modeling results indicated that the intrusion of sea water resulting in a time-lapse microgravity anomalies of 0.12 to 0.18 mGal, at soil layer density of 0.15 g/cm3 to 0.3 g/cm3 and at depth of 30 to 100 m. These imply that the areas experiencing seawater intrusion were Tanjung Mas, SPBE Bandarharjo, Brass, Old Market Boom and Johar as the microgravity measured there were in the range of 0.12 to 0.18 mGal and the density contrast were at 0.15 g/cm3 to 0.28 g/cm3. Areas that experienced fluid reduction were Puri Anjasmoro, Kenconowungu and Puspowarno with microgravity changes from -0.06 mGal to -0.18 mGal.

  19. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

    PubMed

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-02-28

    Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

  20. Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

    PubMed Central

    Schlimpert, Susan; Flärdh, Klas; Buttner, Mark

    2016-01-01

    Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series. PMID:26967231

  1. Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.

    PubMed

    Mathieu, Evelien; Paul, Colin D; Stahl, Richard; Vanmeerbeeck, Geert; Reumers, Veerle; Liu, Chengxun; Konstantopoulos, Konstantinos; Lagae, Liesbet

    2016-08-16

    Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 μm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.

  2. Debye decomposition of time-lapse spectral induced polarisation data

    NASA Astrophysics Data System (ADS)

    Weigand, M.; Kemna, A.

    2016-01-01

    Spectral induced polarisation (SIP) measurements capture the low-frequency electrical properties of soils and rocks and provide a non-invasive means to access lithological, hydrogeological, and geochemical properties of the subsurface. The Debye decomposition (DD) approach is now increasingly being used to analyse SIP signatures in terms of relaxation time distributions due to its flexibility regarding the shape of the spectra. Imaging and time-lapse (monitoring) SIP measurements, capturing SIP variations in space and time, respectively, are now more and more conducted and lead to a drastic increase in the number of spectra considered, which prompts the need for robust and reliable DD tools to extract quantitative parameters from such data. We here present an implementation of the DD method for the analysis of a series of SIP data sets which are expected to only smoothly change in terms of spectral behaviour, such as encountered in many time-lapse applications where measurement geometry does not change. The routine is based on a non-linear least-squares inversion scheme with smoothness constraints on the spectral variation and in addition from one spectrum of the series to the next to deal with the inherent ill-posedness and non-uniqueness of the problem. By means of synthetic examples with typical SIP characteristics we elucidate the influence of the number and range of considered relaxation times on the inversion results. The source code of the presented routines is provided under an open source licence as a basis for further applications and developments.

  3. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle

    PubMed Central

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  4. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

    PubMed

    Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

  5. Automatic segmentation and analysis of fibrin networks in 3D confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Liu, Xiaomin; Mu, Jian; Machlus, Kellie R.; Wolberg, Alisa S.; Rosen, Elliot D.; Xu, Zhiliang; Alber, Mark S.; Chen, Danny Z.

    2012-02-01

    Fibrin networks are a major component of blood clots that provides structural support to the formation of growing clots. Abnormal fibrin networks that are too rigid or too unstable can promote cardiovascular problems and/or bleeding. However, current biological studies of fibrin networks rarely perform quantitative analysis of their structural properties (e.g., the density of branch points) due to the massive branching structures of the networks. In this paper, we present a new approach for segmenting and analyzing fibrin networks in 3D confocal microscopy images. We first identify the target fibrin network by applying the 3D region growing method with global thresholding. We then produce a one-voxel wide centerline for each fiber segment along which the branch points and other structural information of the network can be obtained. Branch points are identified by a novel approach based on the outer medial axis. Cells within the fibrin network are segmented by a new algorithm that combines cluster detection and surface reconstruction based on the α-shape approach. Our algorithm has been evaluated on computer phantom images of fibrin networks for identifying branch points. Experiments on z-stack images of different types of fibrin networks yielded results that are consistent with biological observations.

  6. 3D tracking the Brownian motion of colloidal particles using digital holographic microscopy and joint reconstruction.

    PubMed

    Verrier, Nicolas; Fournier, Corinne; Fournel, Thierry

    2015-06-01

    In-line digital holography is a valuable tool for sizing, locating, and tracking micro- or nano-objects in a volume. When a parametric imaging model is available, inverse problem approaches provide a straightforward estimate of the object parameters by fitting data with the model, thereby allowing accurate reconstruction. As recently proposed and demonstrated, combining pixel super-resolution techniques with inverse problem approaches improves the estimation of particle size and 3D position. Here, we demonstrate the accurate tracking of colloidal particles in Brownian motion. Particle size and 3D position are jointly optimized from video holograms acquired with a digital holographic microscopy setup based on a low-end microscope objective (×20, NA 0.5). Exploiting information redundancy makes it possible to characterize particles with a standard deviation of 15 nm in size and a theoretical resolution of 2×2×5  nm3 for position under additive white Gaussian noise assumption.

  7. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking

    SciTech Connect

    Dettmer, Simon L.; Keyser, Ulrich F.; Pagliara, Stefano

    2014-02-15

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g., electrokinetic or dielectrophoretic forces.

  8. 3D Analysis of Porosity in a Ceramic Coating Using X-ray Microscopy

    NASA Astrophysics Data System (ADS)

    Klement, Uta; Ekberg, Johanna; Kelly, Stephen T.

    2017-02-01

    Suspension plasma spraying (SPS) is a new, innovative plasma spray technique using a feedstock consisting of fine powder particles suspended in a liquid. Using SPS, ceramic coatings with columnar microstructures have been produced which are used as topcoats in thermal barrier coatings. The microstructure contains a wide pore size range consisting of inter-columnar spacings, micro-pores and nano-pores. Hence, determination of total porosity and pore size distribution is a challenge. Here, x-ray microscopy (XRM) has been applied for describing the complex pore space of the coatings because of its capability to image the (local) porosity within the coating in 3D at a resolution down to 50 nm. The possibility to quantitatively segment the analyzed volume allows analysis of both open and closed porosity. For an yttria-stabilized zirconia coating with feathery microstructure, both open and closed porosity were determined and it could be revealed that 11% of the pore volumes (1.4% of the total volume) are closed pores. The analyzed volume was reconstructed to illustrate the distribution of open and closed pores in 3D. Moreover, pore widths and pore volumes were determined. The results on the complex pore space obtained by XRM are discussed in connection with other porosimetry techniques.

  9. Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.

    PubMed

    Lenser, Thorsten; Weisshart, Klaus; Ulbricht, Tobias; Klement, Karolin; Hemmerich, Peter

    2010-01-01

    The three-dimensional (3D) architecture of the cell nucleus is determined not only by the presence of subnuclear domains, such as the nuclear envelope, chromosome territories, and nuclear bodies, but also by smaller domains which form in response to specific functions, such as RNA transcription, DNA replication, and DNA repair. Since both stable and dynamic structures contribute to nuclear morphology, it is important to study the biophysical principles of the formation of macromolecular assemblies within the nucleus. For this purpose, a variety of fluorescence fluctuation microscopy techniques can be applied. Here, we summarize our current knowledge on the 3D architecture of the mammalian cell nucleus and describe in detail how the assembly of functional nuclear protein complexes can be analyzed in living cells using fluorescence bleaching techniques, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and mathematical modeling. In conclusion, the application of all these techniques in combination is a powerful tool to assess the full spectrum of nuclear protein dynamics and to understand the biophysical principles underlying nuclear structure and function.

  10. Automated Atom-By-Atom Three-Dimensional (3D) Reconstruction of Field Ion Microscopy Data.

    PubMed

    Dagan, Michal; Gault, Baptiste; Smith, George D W; Bagot, Paul A J; Moody, Michael P

    2017-03-20

    An automated procedure has been developed for the reconstruction of field ion microscopy (FIM) data that maintains its atomistic nature. FIM characterizes individual atoms on the specimen's surface, evolving subject to field evaporation, in a series of two-dimensional (2D) images. Its unique spatial resolution enables direct imaging of crystal defects as small as single vacancies. To fully exploit FIM's potential, automated analysis tools are required. The reconstruction algorithm developed here relies on minimal assumptions and is sensitive to atomic coordinates of all imaged atoms. It tracks the atoms across a sequence of images, allocating each to its respective crystallographic plane. The result is a highly accurate 3D lattice-resolved reconstruction. The procedure is applied to over 2000 tungsten atoms, including ion-implanted planes. The approach is further adapted to analyze carbides in a steel matrix, demonstrating its applicability to a range of materials. A vast amount of information is collected during the experiment that can underpin advanced analyses such as automated detection of "out of sequence" events, subangstrom surface displacements and defects effects on neighboring atoms. These analyses have the potential to reveal new insights into the field evaporation process and contribute to improving accuracy and scope of 3D FIM and atom probe characterization.

  11. A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy.

    PubMed

    Voss, Neil R; Lyumkis, Dmitry; Cheng, Anchi; Lau, Pick-Wei; Mulder, Anke; Lander, Gabriel C; Brignole, Edward J; Fellmann, Denis; Irving, Christopher; Jacovetty, Erica L; Leung, Albert; Pulokas, James; Quispe, Joel D; Winkler, Hanspeter; Yoshioka, Craig; Carragher, Bridget; Potter, Clinton S

    2010-03-01

    Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.

  12. Analysis of incomplete excisions of basal-cell carcinomas after breadloaf microscopy compared with 3D-microscopy: a prospective randomized and blinded study.

    PubMed

    Boehringer, Alexandra; Adam, Patrick; Schnabl, Saskia; Häfner, Hans-Martin; Breuninger, Helmut

    2015-08-01

    Basal-cell carcinomas may show irregular, asymmetric subclinical growth. This study analyzed the efficacy of 'breadloaf' microscopy (serial sectioning) and three-dimensional (3D) microscopy in detecting positive tumor margins. Two hundred eighty-three (283) tumors (51.2%) were put into the breadloaf microscopy group; 270 tumors (48.8%) into the 3D microscopy group. The position of any detected tumor outgrowths was identified in clock face fashion. The time required for cutting and embedding the specimens and the examination of the microscopic slides was measured. Patient/tumor characteristics and surgical margins did not differ significantly. Tumor outgrowths at the excision margin were found in 62 of 283 cases (21.9%) in the breadloaf microscopy group and in 115 of 270 cases (42.6%) in the 3D microscopy group, constituting a highly significant difference (p < 0.001). This difference held true with incomplete excision of fibrosing (infiltrative/sclerosing/morpheaform) tumors [32.9% in the breadloaf microscopy group and 57.5% in the 3D microscopy group (p = 0.003)] and also with solid (nodular) tumors [16.1 and 34.2%, respectively (p < 0.001)]. The mean overall examination time required showed no important difference. In summary, for detection of tumor outgrowths, 3D microscopy has almost twice the sensitivity of breadloaf microscopy, particularly in the situation of aggressive/infiltrative carcinomas.

  13. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-09-29

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

  14. Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

    PubMed Central

    Liew, Andrew T. F.; Monahan, Leigh G.; Whitchurch, Cynthia B.; Harry, Elizabeth J.

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide. PMID:25286090

  15. Time-lapse three-dimensional inversion of complex conductivity data using an active time constrained (ATC) approach

    USGS Publications Warehouse

    Karaoulis, M.; Revil, A.; Werkema, D.D.; Minsley, B.J.; Woodruff, W.F.; Kemna, A.

    2011-01-01

    Induced polarization (more precisely the magnitude and phase of impedance of the subsurface) is measured using a network of electrodes located at the ground surface or in boreholes. This method yields important information related to the distribution of permeability and contaminants in the shallow subsurface. We propose a new time-lapse 3-D modelling and inversion algorithm to image the evolution of complex conductivity over time. We discretize the subsurface using hexahedron cells. Each cell is assigned a complex resistivity or conductivity value. Using the finite-element approach, we model the in-phase and out-of-phase (quadrature) electrical potentials on the 3-D grid, which are then transformed into apparent complex resistivity. Inhomogeneous Dirichlet boundary conditions are used at the boundary of the domain. The calculation of the Jacobian matrix is based on the principles of reciprocity. The goal of time-lapse inversion is to determine the change in the complex resistivity of each cell of the spatial grid as a function of time. Each model along the time axis is called a 'reference space model'. This approach can be simplified into an inverse problem looking for the optimum of several reference space models using the approximation that the material properties vary linearly in time between two subsequent reference models. Regularizations in both space domain and time domain reduce inversion artefacts and improve the stability of the inversion problem. In addition, the use of the time-lapse equations allows the simultaneous inversion of data obtained at different times in just one inversion step (4-D inversion). The advantages of this new inversion algorithm are demonstrated on synthetic time-lapse data resulting from the simulation of a salt tracer test in a heterogeneous random material described by an anisotropic semi-variogram. ?? 2011 The Authors Geophysical Journal International ?? 2011 RAS.

  16. Streaming level set algorithm for 3D segmentation of confocal microscopy images.

    PubMed

    Gouaillard, Alexandre; Mosaliganti, Kishore; Gelas, Arnaud; Souhait, Lydie; Obholzer, Nikolaus; Megason, Sean

    2009-01-01

    We present a high performance variant of the popular geodesic active contours which are used for splitting cell clusters in microscopy images. Previously, we implemented a linear pipelined version that incorporates as many cues as possible into developing a suitable level-set speed function so that an evolving contour exactly segments a cell/nuclei blob. We use image gradients, distance maps, multiple channel information and a shape model to drive the evolution. We also developed a dedicated seeding strategy that uses the spatial coherency of the data to generate an over complete set of seeds along with a quality metric which is further used to sort out which seed should be used for a given cell. However, the computational performance of any level-set methodology is quite poor when applied to thousands of 3D data-sets each containing thousands of cells. Those data-sets are common in confocal microscopy. In this work, we explore methods to stream the algorithm in shared memory, multi-core environments. By partitioning the input and output using spatial data structures we insure the spatial coherency needed by our seeding algorithm as well as improve drastically the speed without memory overhead. Our results show speed-ups up to a factor of six.

  17. Immuno- and correlative light microscopy-electron tomography methods for 3D protein localization in yeast.

    PubMed

    Mari, Muriel; Geerts, Willie J C; Reggiori, Fulvio

    2014-10-01

    Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three-dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno-electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy-electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.

  18. Quantitative detection of fluid distribution using time-lapse seismic

    NASA Astrophysics Data System (ADS)

    Tsuneyama, Futoshi

    The quantitative evaluation of time-lapse seismic data remains a challenge due to poor match between the model predictions and the actual seismic data. Velocity anisotropy is one important reason for the mismatch. I compile experimental velocity-anisotropy data from cores to explore the empirical relationships between anisotropy parameters and general well-log information. Then, I develop a method to estimate Thomsen's anisotropy parameters ε and gamma using a regression of the data in the crossplot domain between velocity and porosity. I present an application result of the method to demonstrate the significance of the correction. Next, using the corrected velocity, I present a method of impedance decomposition into Vp, Vs, and rho using three elastic impedances derived from the seismic inversion of angle stacks. In general, the maximum stack angle of seismic data is limited to be less than 30°, which is not wide enough to obtain the stable calculation result. I discuss the effect of noise on the analysis as the most important reason that decomposition is difficult. I show an innovative method incorporating rock-physics bounds as constraints for the analysis. I apply it to an actual dataset from an offshore oil field; I demonstrate the result of analysis for sand-body detection. Based on the estimated Vp, V s, rho and shale volume from the elastic impedances, I develop a workflow to determine the saturation of formation-water, oil and gas from seismic data. First, I consider the pressure effect and the saturation scale of fluids for time-lapse seismic analysis. Second, I demonstrate a deterministic approach to computing the fluid saturation to evaluate time-lapse seismic data. In this approach, I derive the physical properties of the water-saturated sandstone reservoir. Then, by comparing the in-situ-fluid-saturated properties with the 100% formation-water-saturated reservoir properties, I determine the bulk modulus and the density of the fluid phase in the

  19. Time-Lapse Evaluation of Interactions Between Biodegradable Mg Particles and Cells.

    PubMed

    Alvarez, Florencia; Lozano Puerto, Rosa M; Pérez-Maceda, Blanca; Grillo, Claudia A; Fernández Lorenzo de Mele, Mónica

    2016-02-01

    Mg-based implants have promising applications as biodegradable materials in medicine for orthopedic, dental, and cardiovascular therapies. During wear and degradation microdebris are released. Time-lapse multidimensional microscopy (MM) is proposed here as a suitable tool to follow, in fixed intervals over 24-h periods, the interaction between cells and particles. Results of MM show interactions of macrophages (J774) with the magnesium particles (MgPa) that led to modifications of cell size and morphology, a decrease in duplication rate, and cell damage. Corrosion products were progressively formed on the surface of the particles and turbulence was generated due to hydrogen development. Changes were more significant after treating MgPa with potassium fluoride. In order to complement MM observations, membrane damage as detected by a lactase dehydrogenase (LDH) assay and mitochondrial activity as detected by a WST-1 assay with macrophages and osteoblasts (MC3T3-E1) were compared. A more significant concentration-dependent effect was detected for macrophages exposed to MgPa than for osteoblasts. Accordingly, complementary data showed that viability and cell cycle seem to be more altered in macrophages. In addition, protein profiles and expression of proteins associated with the adhesion process changed in the presence of MgPa. These studies revealed that time-lapse MM is a helpful tool for monitoring changes of biodegradable materials and the biological surrounding in real time and in situ. This information is useful in studies related to biodegradable biomaterials.

  20. Time-lapse video sysem used to study nesting gyrfalcons

    USGS Publications Warehouse

    Booms, Travis; Fuller, Mark R.

    2003-01-01

    We used solar-powered time-lapse video photography to document nesting Gyrfalcon (Falco rusticolus) food habits in central West Greenland from May to July in 2000 and 2001. We collected 2677.25 h of videotape from three nests, representing 94, 87, and 49% of the nestling period at each nest. The video recorded 921 deliveries of 832 prey items. We placed 95% of the items into prey categories. The image quality was good but did not reveal enough detail to identify most passerines to species. We found no evidence that Gyrfalcons were negatively affected by the video system after the initial camera set-up. The video system experienced some mechanical problems but proved reliable. The system likely can be used to effectively document the food habits and nesting behavior of other birds, especially those delivering large prey to a nest or other frequently used site.

  1. Time-Lapse Geophysics for Aquifer Characterization and Remediation Monitoring

    NASA Astrophysics Data System (ADS)

    Lane, J. W.

    2003-12-01

    Time-lapse monitoring of subsurface processes is an emerging and promising area of hydrogeophysics. The use of non-invasive or minimally invasive geophysical methods to indirectly measure time-varying fluid saturation, solute concentration, and other hydraulic and geochemical conditions facilitates cost-effective aquifer characterization and remediation. The USGS Office of Ground Water, Branch of Geophysics, in cooperation with USEPA, DOD, and university researchers, has applied time-lapse geophysics for site characterization and remediation monitoring at a number of sites. This talk presents recent examples of applied research, including: (1) application of cross-borehole and surface-to-borehole radar methods to monitor vegetable-oil emulsion injections for biostimulation at a Navy site in Fridley, MN; (2) application of borehole and cross-borehole radar methods to monitor steam injections for remediation of VOCs at the former Loring Air Force Base, ME; (3) application of electrical resistivity tomography to monitor saline tracer tests at the Massachusetts Military Reservation, MA; (4) use of borehole and cross-borehole flowmeter and a discrete-zone packer system to characterize bedrock aquifer hydraulics and water quality at the University of Connecticut landfill, Storrs, CT; and (5) application of crosshole radar methods to monitor a saline tracer in fractured bedrock at the USGS Mirror Lake Site, NH. The goals of these studies are (1) to provide increasingly quantitative information about the subsurface, critical for developing models of aquifer structure, dynamics, and processes, and (2) identification of the spatial and temporal distributions of tracers, contamination, and fluids injected to enhance degradation of contaminants.

  2. Dust Storm Time Lapse Shows Opportunity's Skies Darken

    NASA Technical Reports Server (NTRS)

    2007-01-01

    [figure removed for brevity, see original site] Dust Storm Time Lapse Shows Opportunity's Skies Darken

    NASA's Opportunity rover is literally seeing some of its darkest days. Both Mars Exploration Rovers have been riding out a regional dust storm for several weeks. Conditions became particularly dreary in the Meridiani Planum region where Opportunity sits, perched on the edge of 'Victoria Crater.'

    This image is a time-lapse composite where each horizon-survey image has been compressed horizontally (but not vertically) to emphasize the sky. The relative brightness and darkness of the sky from sol to sol (over a 30-sol period beginning June 14, 2007) is depicted accurately in these images, which view roughly the same part of the plains southwest of the rover. The images are approximately true color composites, generated from calibrated radiance data files using the panoramic camera's 601-nanometer, 535-nanometer and 482-nanometer filters.

    The rovers' atmospheric science team is concerned that smaller, regional dust storms could expand into a larger, globe-encircling storm. That could extend the time the sun stays obscured, challenging the capability of Opportunity's solar panels to produce enough electricity for the rover to function.

    Fortunately, as of July 19, 2007, the Opportunity site is clearing slightly. When the storm ends, atmospheric scientists hope to review data from the rovers that will help them determine what sort of dust was being lifted and distributed.

    The numbers across the top of the image report a measurement of atmospheric opacity, called by the Greek letter tau. The lower the number, the clearer the sky. Both Opportunity and Spirit have been recording higher tau measurements in July 2007 than they had seen any time previously in their three and a half years on Mars. The five sol numbers across the bottom correspond (left to right) to June 14, June 30, July 5, July 13 and July 15, 2007.

  3. Confocal laser scanning microscopy and 3-D reconstructions of neuronal structures in human brain cortex.

    PubMed

    Belichenko, P V; Dahlström, A

    1995-09-01

    Human brain material was studied with Lucifer yellow (LY) microinjections, indirect Texas red immunofluorescence, and confocal laser scanning microscopy (CLSM). The scanned images were transferred to a Silicon Graphics (SG) IRIS computer equipped with software for reconstructing the 3-D architecture of cells. By employing dual channel CLSM (Bio-Rad MRC 600), LY-injected cells and Texas red immunofluorescence could be studied simultaneously. Autopsy material with 2- to 48-h postmortem delays (6 control and 2 Rett's syndrome cases) as well as biopsy material (14 cases with therapy-resistant partial epilepsy--TRPE--undergoing neurosurgery) were used. In each specimen, 100-200 pyramidal and nonpyramidal neurons were visualized by LY microinjection. Single neurons were imaged and 2-D reconstructions of each neuron were made using z-projections of serial optical images; 3-D reconstructions and rotations were computed using the SG workstation, with VoxelView software from Vital Images (UK), and stored in a "neuronal library" on laser or magnetic optical disks. In Ret's syndrome cases and in patients with TRPE various abnormalities in the dendritic geometry of pyramidal and nonpyramidal cells have been found. The combination of LY injections with immunofluorescence allows the investigation of transmitter-related substances around the LY-injected cells. Using antibodies to synaptic vesicle proteins, presynaptic elements docking onto individual spines have been demonstrated. This approach may contribute to the understanding of different neurological and psychiatric disorders and may be useful in the Mapping of the Human Brain project. It may also be integrated with functional imaging by PET scan and with the human genome project.

  4. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  5. Single particle cryo-electron microscopy and 3-D reconstruction of viruses.

    PubMed

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3-4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

  6. A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development

    PubMed Central

    Harris, Kristen M.; Spacek, Josef; Bell, Maria Elizabeth; Parker, Patrick H.; Lindsey, Laurence F.; Baden, Alexander D.; Vogelstein, Joshua T.; Burns, Randal

    2015-01-01

    Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50–60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm3) surrounding a large dendritic spine, a cylinder (~43 μm3) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm3) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1). PMID:26347348

  7. Clean localization super-resolution microscopy for 3D biological imaging

    NASA Astrophysics Data System (ADS)

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-01

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  8. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    NASA Astrophysics Data System (ADS)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  9. Jamming of a soft granular system of hollow elastic shells in 3D using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Jose, Jissy; van Blaaderen, Alfons; Imhof, Arnout

    2014-03-01

    We introduce a new system for jammed matter research consisting of monodisperse, fluorescent, hollow deformable shells, dispersed in an index matched solvent. The interesting fact about these elastic shells is that they undergo buckling: in each contact one of the shells receives an indentation from its neighbor under compressive stress. This kind of deformation is different from the soft granular systems experimentally studied so far like photo elastic disks, emulsions and foams, where the particles are flattened in the region of contact and conserve their volume. Using confocal microscopy and image analysis routines (ImageJ software) we identified the 3D position of the particles with sub pixel resolution. The force law to find the contact forces between pairs of particle is derived from the theory of elasticity of thin shells, where force is proportional to the square root of indentation depth. The distribution of normalized contact forces showed a similar trend like other jammed systems with a peak around the mean and a tail that decayed faster than exponential away from jamming threshold. Further, we also investigated the structure of the jammed packings and contact number distribution with distance to jamming.

  10. Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

    PubMed Central

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced. PMID:24357374

  11. Clean localization super-resolution microscopy for 3D biological imaging

    SciTech Connect

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-15

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  12. Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

    PubMed

    Rolland, Jannick P; Canavesi, Cristina; Tankam, Patrice; Cogliati, Andrea; Lanis, Mara; Santhanam, Anand P

    2016-01-01

    Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 μm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented.

  13. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  14. Model-based segmentation and quantification of subcellular structures in 2D and 3D fluorescent microscopy images

    NASA Astrophysics Data System (ADS)

    Wörz, Stefan; Heinzer, Stephan; Weiss, Matthias; Rohr, Karl

    2008-03-01

    We introduce a model-based approach for segmenting and quantifying GFP-tagged subcellular structures of the Golgi apparatus in 2D and 3D microscopy images. The approach is based on 2D and 3D intensity models, which are directly fitted to an image within 2D circular or 3D spherical regions-of-interest (ROIs). We also propose automatic approaches for the detection of candidates, for the initialization of the model parameters, and for adapting the size of the ROI used for model fitting. Based on the fitting results, we determine statistical information about the spatial distribution and the total amount of intensity (fluorescence) of the subcellular structures. We demonstrate the applicability of our new approach based on 2D and 3D microscopy images.

  15. Time-lapse cinematography in living Drosophila tissues: preparation of material.

    PubMed

    Davis, Ilan; Parton, Richard M

    2006-11-01

    The fruit fly, Drosophila melanogaster, has been an extraordinarily successful model organism for studying the genetic basis of development and evolution. It is arguably the best-understood complex multicellular model system, owing its success to many factors. Recent developments in imaging techniques, in particular sophisticated fluorescence microscopy methods and equipment, now allow cellular events to be studied at high resolution in living material. This ability has enabled the study of features that tend to be lost or damaged by fixation, such as transient or dynamic events. Although many of the techniques of live cell imaging in Drosophila are shared with the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties in keeping the cells alive, introducing fluorescent probes, and imaging through thick hazy cytoplasm. This protocol outlines the preparation of major tissue types amenable to study by time-lapse cinematography and different methods for keeping them alive.

  16. Wavelet compression of three-dimensional time-lapse biological image data.

    PubMed

    Stefansson, H Narfi; Eliceiri, Kevin W; Thomas, Charles F; Ron, Amos; DeVore, Ron; Sharpley, Robert; White, John G

    2005-02-01

    The use of multifocal-plane, time-lapse recordings of living specimens has allowed investigators to visualize dynamic events both within ensembles of cells and individual cells. Recordings of such four-dimensional (4D) data from digital optical sectioning microscopy produce very large data sets. We describe a wavelet-based data compression algorithm that capitalizes on the inherent redunancies within multidimensional data to achieve higher compression levels than can be obtained from single images. The algorithm will permit remote users to roam through large 4D data sets using communication channels of modest bandwidth at high speed. This will allow animation to be used as a powerful aid to visualizing dynamic changes in three-dimensional structures.

  17. In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath.

    PubMed

    Gonzalez, Sergio; Fernando, Ruani; Berthelot, Jade; Perrin-Tricaud, Claire; Sarzi, Emmanuelle; Chrast, Roman; Lenaers, Guy; Tricaud, Nicolas

    2015-07-01

    The myelin sheath that covers a large amount of neurons is critical for their homeostasis, and myelinating glia mitochondria have recently been shown to be essential for neuron survival. However morphological and physiological properties of these organelles remain elusive. Here we report a method to analyze mitochondrial dynamics and morphology in myelinating Schwann cells of living mice using viral transduction and time-lapse multiphoton microscopy. We describe the distribution, shape, size and dynamics of mitochondria in live cells. We also report mitochondrial alterations in Opa1(delTTAG) mutant mice cells at presymptomatic stages, suggesting that mitochondrial defects in myelin contribute to OPA1 related neuropathy and represent a biomarker for the disease.

  18. Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

    PubMed Central

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-01-01

    Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens. PMID:25185543

  19. Nanoscale Nucleosome Dynamics Assessed with Time-lapse AFM

    PubMed Central

    Lyubchenko, Yuri L.

    2013-01-01

    A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as it was once believed. Direct data is required in order to understand the dynamics of nucleosomes more clearly and answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics. PMID:24839467

  20. A spherical harmonics intensity model for 3D segmentation and 3D shape analysis of heterochromatin foci.

    PubMed

    Eck, Simon; Wörz, Stefan; Müller-Ott, Katharina; Hahn, Matthias; Biesdorf, Andreas; Schotta, Gunnar; Rippe, Karsten; Rohr, Karl

    2016-08-01

    The genome is partitioned into regions of euchromatin and heterochromatin. The organization of heterochromatin is important for the regulation of cellular processes such as chromosome segregation and gene silencing, and their misregulation is linked to cancer and other diseases. We present a model-based approach for automatic 3D segmentation and 3D shape analysis of heterochromatin foci from 3D confocal light microscopy images. Our approach employs a novel 3D intensity model based on spherical harmonics, which analytically describes the shape and intensities of the foci. The model parameters are determined by fitting the model to the image intensities using least-squares minimization. To characterize the 3D shape of the foci, we exploit the computed spherical harmonics coefficients and determine a shape descriptor. We applied our approach to 3D synthetic image data as well as real 3D static and real 3D time-lapse microscopy images, and compared the performance with that of previous approaches. It turned out that our approach yields accurate 3D segmentation results and performs better than previous approaches. We also show that our approach can be used for quantifying 3D shape differences of heterochromatin foci.

  1. Infrared differential interference contrast microscopy for overlay metrology on 3D-interconnect bonded wafers

    NASA Astrophysics Data System (ADS)

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-04-01

    Overlay metrology for stacked layers will be playing a key role in bringing 3D IC devices into manufacturing. However, such bonded wafer pairs present a metrology challenge for optical microscopy tools by the opaque nature of silicon. Using infrared microscopy, silicon wafers become transparent to the near-infrared (NIR) wavelengths of the electromagnetic spectrum, enabling metrology at the interface of bonded wafer pairs. Wafers can be bonded face to face (F2F) or face to back (F2B) which the stacking direction is dictated by how the stacks are carried in the process and functionality required. For example, Memory stacks tend to use F2B stacking enables a better managed design. Current commercial tools use single image technique for F2F bonding overlay measurement because depth of focus is sufficient to include both surfaces; and use multiple image techniques for F2B overlay measurement application for the depth of focus is no longer sufficient to include both stacked wafer surfaces. There is a need to specify the Z coordinate or stacking wafer number through the silicon when visiting measurement wafer sites. Two shown images are of the same (X, Y) but separate Z location acquired at focus position of each wafer surface containing overlay marks. Usually the top surface image is bright and clear; however, the bottom surface image is somewhat darker and noisier as an adhesive layer is used in between to bond the silicon wafers. Thus the top and bottom surface images are further processed to achieve similar brightness and noise level before merged for overlay measurement. This paper presents a special overlay measurement technique, using the infrared differential interference contrast (DIC) microscopy technique to measure the F2B wafer bonding overlay by a single shot image. A pair of thinned wafers at 50 and 150 μm thickness is bonded on top of a carrier wafer to evaluate the bonding overlay. It works on the principle of interferometry to gain information about the

  2. Examination of heterogeneous crossing sequences between toner and rollerball pen strokes by digital microscopy and 3-D laser profilometry.

    PubMed

    Montani, Isabelle; Mazzella, Williams; Guichard, Marion; Marquis, Raymond

    2012-07-01

    The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner.

  3. Exploring Time-Lapse Photography as a Means for Qualitative Data Collection

    ERIC Educational Resources Information Center

    Persohn, Lindsay

    2015-01-01

    Collecting information via time-lapse photography is nothing new. Scientists and artists have been using this kind of data since the late 1800s. However, my research and experiments with time-lapse have shown that great potential may lie in its application to educational and social scientific research methods. This article is part history, part…

  4. 3D Quantitative Confocal Laser Microscopy of Ilmenite Volume Distribution in Alpe Arami Olivine

    NASA Astrophysics Data System (ADS)

    Bozhilov, K. N.

    2001-12-01

    The deep origin of the Alpe Arami garnet lherzolite massif in the Swiss Alps proposed by Dobrzhinetskaya et al. (Science, 1996) has been a focus of heated debate. One of the lines of evidence supporting an exhumation from more than 200 km depth includes the abundance, distribution, and orientation of magnesian ilmenite rods in the oldest generation of olivine. This argument has been disputed in terms of the abundance of ilmenite and consequently the maximum TiO2 content in the discussed olivine. In order to address this issue, we have directly measured the volume fraction of ilmenite of the oldest generation of olivine by applying confocal laser scanning microscopy (CLSM). CLSM is a method which allows for three-dimensional imaging and quantitative volume determination by optical sectioning of the objects. The images for 3D reconstruction and measurements were acquired from petrographic thin sections in reflected laser light with 488 nm wavelength. Measurements of more than 80 olivine grains in six thin sections of our material yielded an average volume fraction of 0.31% ilmenite in the oldest generation of olivine from Alpe Arami. This translates into 0.23 wt.% TiO2 in olivine with error in determination of ±0.097 wt.%, a value significantly different from that of 0.02 to 0.03 wt.% TiO2 determined by Hacker et al. (Science, 1997) by a broad-beam microanalysis technique. During the complex geological history of the Alpe Arami massif, several events of metamorphism are recorded which all could have caused increased mobility of the mineral components. Evidence for loss of TiO2 from olivine is the tendency for high densities of ilmenite to be restricted to cores of old grains, the complete absence of ilmenite inclusions from the younger, recrystallized, generation of olivine, and reduction in ilmenite size and abundance in more serpentinized specimens. These observations suggest that only olivine grains with the highest concentrations of ilmenite are close to the

  5. Estimation of atmospheric parameters from time-lapse imagery

    NASA Astrophysics Data System (ADS)

    McCrae, Jack E.; Basu, Santasri; Fiorino, Steven T.

    2016-05-01

    A time-lapse imaging experiment was conducted to estimate various atmospheric parameters for the imaging path. Atmospheric turbulence caused frame-to-frame shifts of the entire image as well as parts of the image. The statistics of these shifts encode information about the turbulence strength (as characterized by Cn2, the refractive index structure function constant) along the optical path. The shift variance observed is simply proportional to the variance of the tilt of the optical field averaged over the area being tracked. By presuming this turbulence follows the Kolmogorov spectrum, weighting functions can be derived which relate the turbulence strength along the path to the shifts measured. These weighting functions peak at the camera and fall to zero at the object. The larger the area observed, the more quickly the weighting function decays. One parameter we would like to estimate is r0 (the Fried parameter, or atmospheric coherence diameter.) The weighting functions derived for pixel sized or larger parts of the image all fall faster than the weighting function appropriate for estimating the spherical wave r0. If we presume Cn2 is constant along the path, then an estimate for r0 can be obtained for each area tracked, but since the weighting function for r0 differs substantially from that for every realizable tracked area, it can be expected this approach would yield a poor estimator. Instead, the weighting functions for a number of different patch sizes can be combined through the Moore-Penrose pseudo-inverse to create a new weighting function which yields the least-squares optimal linear combination of measurements for estimation of r0. This approach is carried out, and it is observed that this approach is somewhat noisy because the pseudo-inverse assigns weights much greater than one to many of the observations.

  6. Liquid crystal lens array for 3D microscopy and endoscope application

    NASA Astrophysics Data System (ADS)

    Huang, Yi-Pai; Hsieh, Po-Yuan; Hassanfiroozi, Amir; Chu, Chao-Yu; Hsuan, Yun; Martinez, Manuel; Javidi, Bahram

    2016-06-01

    In this paper, we demonstrate two liquid crystal (LC) lens array devices for 3D microscope and 3D endoscope applications respectively. Compared with the previous 3D biomedical system, the proposed LC lens arrays are not only switchable between 2D and 3D modes, but also are able to adjust focus in both modes. The multi-function liquid crystal lens (MFLC-lens) array with dual layer electrode has diameter 1.42 mm, which is much smaller than the conventional 3D endoscope with double fixed lenses. The hexagonal liquid crystal micro-lens array (HLC-MLA) instead of fixed micro-lens array in 3D light field microscope can extend the effective depth of field from 60 um to 780 um. To achieve the LC lens arrays, a high-resistance layer needs to be coated on the electrodes to generate an ideal gradient electric-field distribution, which can induce a lens-like form of LC molecules. The parameters and characteristics of high-resistance layer are investigated and discussed with an aim to optimize the performance of liquid crystal lens arrays.

  7. Oil Sands Characteristics and Time-Lapse and P-SV Seismic Steam Monitoring, Athabasca, Canada

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Nakayama, T.; Kashihara, K.; Skinner, L.; Kato, A.

    2008-12-01

    -injection. The differences of the seismic responses between the time-lapse seismic volumes can be quantitatively explained by P-wave velocity decrease of the oil sands layers due to steam-injection. In addition, the data suggests that a larger area would be influenced by pressure than temperature. We calculate several seismic attributes such as RMS values of amplitude difference, maximum cross correlations, and interval velocity differences. These attributes are integrated by using self-organization maps (SOM) and K-means methods. By this analysis, we are able to distinguish areas of steam chamber growth from transitional and non-affected areas. In addition, 3D P-SV converted-wave processing and analysis are applied on the second 3D data set (recorded with three-component digital sensor). Low Vp/Vs values in the P-SV volume show areas of steam chamber development, and high Vp/Vs values indicate transitional zones. Our analysis of both time-lapse 3D seismic and 3D P-SV data along with the rock physics model can be used to monitor qualitatively and quantitatively the rock property changes of the inter-well reservoir sands in the field.

  8. A virtually imaged defocused array (VIDA) for high-speed 3D microscopy.

    PubMed

    Schonbrun, Ethan; Di Caprio, Giuseppe

    2016-10-01

    We report a method to capture a multifocus image stack based on recording multiple reflections generated by imaging through a custom etalon. The focus stack is collected in a single camera exposure and consequently the information needed for 3D reconstruction is recorded in the camera integration time, which is only 100 µs. We have used the VIDA microscope to temporally resolve the multi-lobed 3D morphology of neutrophil nuclei as they rotate and deform through a microfluidic constriction. In addition, we have constructed a 3D imaging flow cytometer and quantified the nuclear morphology of nearly a thousand white blood cells flowing at a velocity of 3 mm per second. The VIDA microscope is compact and simple to construct, intrinsically achromatic, and the field-of-view and stack number can be easily reconfigured without redesigning diffraction gratings and prisms.

  9. Fluorescent stereo microscopy for 3D surface profilometry and deformation mapping.

    PubMed

    Hu, Zhenxing; Luo, Huiyang; Du, Yingjie; Lu, Hongbing

    2013-05-20

    Recently, mechanobiology has received increased attention. For investigation of biofilm and cellular tissue, measurements of the surface topography and deformation in real-time are a pre-requisite for understanding the growth mechanisms. In this paper, a novel three-dimensional (3D) fluorescent microscopic method for surface profilometry and deformation measurements is developed. In this technique a pair of cameras are connected to a binocular fluorescent microscope to acquire micrographs from two different viewing angles of a sample surface doped or sprayed with fluorescent microparticles. Digital image correlation technique is used to search for matching points in the pairing fluorescence micrographs. After calibration of the system, the 3D surface topography is reconstructed from the pair of planar images. When the deformed surface topography is compared with undeformed topography using fluorescent microparticles for movement tracking of individual material points, the full field deformation of the surface is determined. The technique is demonstrated on topography measurement of a biofilm, and also on surface deformation measurement of the biofilm during growth. The use of 3D imaging of the fluorescent microparticles eliminates the formation of bright parts in an image caused by specular reflections. The technique is appropriate for non-contact, full-field and real-time 3D surface profilometry and deformation measurements of materials and structures at the microscale.

  10. Imaging bacterial 3D motion using digital in-line holographic microscopy and correlation-based de-noising algorithm

    PubMed Central

    Molaei, Mehdi; Sheng, Jian

    2014-01-01

    Abstract: Better understanding of bacteria environment interactions in the context of biofilm formation requires accurate 3-dimentional measurements of bacteria motility. Digital Holographic Microscopy (DHM) has demonstrated its capability in resolving 3D distribution and mobility of particulates in a dense suspension. Due to their low scattering efficiency, bacteria are substantially difficult to be imaged by DHM. In this paper, we introduce a novel correlation-based de-noising algorithm to remove the background noise and enhance the quality of the hologram. Implemented in conjunction with DHM, we demonstrate that the method allows DHM to resolve 3-D E. coli bacteria locations of a dense suspension (>107 cells/ml) with submicron resolutions (<0.5 µm) over substantial depth and to obtain thousands of 3D cell trajectories. PMID:25607177

  11. Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging

    PubMed Central

    Boulanger, Jérôme; Gueudry, Charles; Münch, Daniel; Cinquin, Bertrand; Paul-Gilloteaux, Perrine; Bardin, Sabine; Guérin, Christophe; Senger, Fabrice; Blanchoin, Laurent; Salamero, Jean

    2014-01-01

    Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second. PMID:25404337

  12. Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

    PubMed

    Boulanger, Jérôme; Gueudry, Charles; Münch, Daniel; Cinquin, Bertrand; Paul-Gilloteaux, Perrine; Bardin, Sabine; Guérin, Christophe; Senger, Fabrice; Blanchoin, Laurent; Salamero, Jean

    2014-12-02

    Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

  13. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy

    PubMed Central

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L.; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-01-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  14. Biomarkers identified with time-lapse imaging: discovery, validation, and practical application

    PubMed Central

    Chen, Alice A.; Tan, Lei; Suraj, Vaishali; Pera, Renee Reijo; Shen, Shehua

    2014-01-01

    Time-lapse markers,” which are defined by time-lapse imaging and correlated with clinical outcomes, may provide embryologists with new opportunities for improving embryo selection. This article provides an overview of noninvasive biomarkers defined by time-lapse imaging studies. In addition to comprehensively reviewing the discovery of each time-lapse marker, it focuses on the criteria necessary for their successful integration into clinical practice, including [1] statistical and biological significance, [2] validation through prospective clinical studies, and [3] development of reliable technology to measure and quantify the time-lapse marker. Because manual analysis of time-lapse images is labor intensive and limits the practical use of the image data in the clinic, automated image analysis software platforms may contribute substantially to improvements in embryo selection accuracy. Ultimately, time-lapse markers that are based on a foundation of basic research, validated through prospective clinical studies, and enabled by a reliable quantification technology may improve IVF success rates, encourage broader adoption of single-embryo transfer, and reduce the risks associated with multiple gestation pregnancies. PMID:23499001

  15. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB☆

    PubMed Central

    Lagerstedt, Ingvar; Moore, William J.; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R.; Kleywegt, Gerard J.

    2013-01-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed. PMID:24113529

  16. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB.

    PubMed

    Lagerstedt, Ingvar; Moore, William J; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R; Kleywegt, Gerard J

    2013-11-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed.

  17. Potential and limitations of microscopy and Raman spectroscopy for live-cell analysis of 3D cell cultures.

    PubMed

    Charwat, Verena; Schütze, Karin; Holnthoner, Wolfgang; Lavrentieva, Antonina; Gangnus, Rainer; Hofbauer, Pablo; Hoffmann, Claudia; Angres, Brigitte; Kasper, Cornelia

    2015-07-10

    Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell

  18. Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures.

    PubMed

    Chen, Yong; Cai, Jiye; Zhao, Tao; Wang, Chenxi; Dong, Shuo; Luo, Shuqian; Chen, Zheng W

    2005-06-01

    The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.

  19. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    PubMed

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  20. Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

    PubMed

    Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

    2011-07-01

    Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults.

  1. Time-lapse imaging of neural development: Zebrafish lead the way into the fourth dimension

    PubMed Central

    Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

    2011-01-01

    Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. PMID:21305690

  2. 3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

    PubMed Central

    Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

    2016-01-01

    Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

  3. 3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

    PubMed

    Limandri, Silvina; Galván Josa, Víctor; Valentinuzzi, María Cecilia; Chena, María Emilia; Castellano, Gustavo

    2016-05-01

    The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

  4. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

    PubMed Central

    Arnold, Jan; Mahamid, Julia; Lucic, Vladan; de Marco, Alex; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony A.; Baumeister, Wolfgang; Plitzko, Jürgen M.

    2016-01-01

    The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy. PMID:26769364

  5. Multi-modal digital holographic microscopy for wide-field fluorescence and 3D phase imaging

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Xia, Peng; Matoba, Osamu; Nitta, Koichi; Awatsuji, Yasuhiro

    2016-03-01

    Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

  6. Sample holder for axial rotation of specimens in 3D microscopy.

    PubMed

    Bruns, T; Schickinger, S; Schneckenburger, H

    2015-10-01

    In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

  7. Orb-2's Antares Rolls Out to Launch Pad (Time-Lapse)

    NASA Video Gallery

    This time-lapse video shows the roll out of the Orbital Sciences Corporation Antares rocket, with the Cygnus spacecraft aboard, from the Horizontal Integration Facility to the Mid-Atlantic Regional...

  8. NASA HS3 Mission Time Lapse Highlights Cameras Over Tropical Systems

    NASA Video Gallery

    This is a time-lapse video created with images recorded during a recent HS3 Science Missions with the NASA Global Hawk. Shown are the images from the Daylight, HDVis, and the Low-light Cameras on t...

  9. Time-Lapse of Backplane of the JWST Being Moved Into Clean Room

    NASA Video Gallery

    This is a time-lapse video of the center section of the 'pathfinder' backplane for NASA's James Webb Space Telescope being moved into the clean room at NASA's Goddard Space Flight Center in Greenbe...

  10. Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

    NASA Astrophysics Data System (ADS)

    Chen, Szu-Yu; Hsu, Yu John; Yeh, Chia-Hua; Chen, S.-Wei; Chung, Chien-Han

    2015-03-01

    A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

  11. Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

    PubMed

    Ancin, H; Roysam, B; Dufresne, T E; Chestnut, M M; Ridder, G M; Szarowski, D H; Turner, J N

    1996-11-01

    Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

  12. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  13. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  14. Analysis of thin baked-on silicone layers by FTIR and 3D-Laser Scanning Microscopy.

    PubMed

    Funke, Stefanie; Matilainen, Julia; Nalenz, Heiko; Bechtold-Peters, Karoline; Mahler, Hanns-Christian; Friess, Wolfgang

    2015-10-01

    Pre-filled syringes (PFS) and auto-injection devices with cartridges are increasingly used for parenteral administration. To assure functionality, silicone oil is applied to the inner surface of the glass barrel. Silicone oil migration into the product can be minimized by applying a thin but sufficient layer of silicone oil emulsion followed by thermal bake-on versus spraying-on silicone oil. Silicone layers thicker than 100nm resulting from regular spray-on siliconization can be characterized using interferometric profilometers. However, the analysis of thin silicone layers generated by bake-on siliconization is more challenging. In this paper, we have evaluated Fourier transform infrared (FTIR) spectroscopy after solvent extraction and a new 3D-Laser Scanning Microscopy (3D-LSM) to overcome this challenge. A multi-step solvent extraction and subsequent FTIR spectroscopy enabled to quantify baked-on silicone levels as low as 21-325μg per 5mL cartridge. 3D-LSM was successfully established to visualize and measure baked-on silicone layers as thin as 10nm. 3D-LSM was additionally used to analyze the silicone oil distribution within cartridges at such low levels. Both methods provided new, highly valuable insights to characterize the siliconization after processing, in order to achieve functionality.

  15. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy

    PubMed Central

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-01-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  16. 3D myofibril imaging in live cardiomyocytes via hybrid SHG-TPEF microscopy

    NASA Astrophysics Data System (ADS)

    Shao, Yonghong; Liu, Honghai; Ye, Tong; Borg, Tom; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce

    2011-03-01

    We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG and TPEF in the research of myofibrillogensis.

  17. Web-based volume slicer for 3D electron-microscopy data from EMDB.

    PubMed

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J; Patwardhan, Ardan

    2016-05-01

    We describe the functionality and design of the Volume slicer - a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale.

  18. 3D-confocal microscopy for surface analysis of microstructured materials

    NASA Astrophysics Data System (ADS)

    Kagerer, Bernd; Brodmann, Rainer; Valentin, Juergen; Filzek, Jan; Popp, Uwe

    2002-06-01

    The surface of technical materials is playing an ever more important part in modern production processes. However, standard roughness values, which are obtained from a profile, frequently no longer provide sufficient descriptions. What are desired are three-dimensional measurements of surfaces over a macroscopic range with a high degree of vertical and lateral resolution. This has become necessary to be able to describe both deterministic and non-deterministic structures in the same fashion. Due to increased requirements for data and the measuring speed demanded by industry, only optical systems are a possibility. Using the example of tribology, the capability of this technology is shown in this article on the basis of the commercial confocal 3D white light microscope, the NanoFocusTMμSurfTM. On the one hand, the technology and data preparation used are discussed, and on the other, a comparison is drawn with other standard optical measuring methods.

  19. Fast 3-D temporal focusing microscopy using an electrically tunable lens.

    PubMed

    Jiang, Jun; Zhang, Dapeng; Walker, Steven; Gu, Chenglin; Ke, Ya; Yung, Wing Ho; Chen, Shih-chi

    2015-09-21

    In this paper, we present a 3-D temporal focusing microscope based on an electrically tunable lens (ETL) and a femtosecond regenerative laser amplifier. The focus-tunable lens provides a fast and compact way to perform non-mechanical z-scanning and resolves the blurry image issue compared with GVD-based z-scanning methods. The optical performance of the temporal focusing system, including z-scanning characteristics, the associated the magnification variation, and the lateral and axial resolution, has been studied and characterized using calibrated Rhodamine-6G thin film sample, fluorescent beads, and pollen samples. Lastly, we demonstrate the optical cross-sectioning and z-scanning capability with an in vivo experiment, where Ca(2+) imaging of neurons in GaCamp6 labeled zebrafish was performed.

  20. Uncertainty studies of topographical measurements on steel surface corrosion by 3D scanning electron microscopy.

    PubMed

    Kang, K W; Pereda, M D; Canafoglia, M E; Bilmes, P; Llorente, C; Bonetto, R

    2012-02-01

    Pitting corrosion is a damage mechanism quite serious and dangerous in both carbon steel boiler tubes for power plants which are vital to most industries and stainless steels for orthopedic human implants whose demand, due to the increase of life expectation and rate of traffic accidents, has sharply increased. Reliable methods to characterize this kind of damage are becoming increasingly necessary, when trying to evaluate the advance of damage and to establish the best procedures for component inspection in order to determine remaining lives and failure mitigation. A study about the uncertainties on the topographies of corrosion pits from 3D SEM images, obtained at low magnifications (where errors are greater) and different stage tilt angles were carried out using an in-house software previously developed. Additionally, measurements of pit depths on biomaterial surfaces, subjected to two different surface treatments on stainless steels, were carried out. The different depth distributions observed were in agreement with electrochemical measurements.

  1. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

    PubMed

    Xu, Jianquan; Tehrani, Kayvan F; Kner, Peter

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.

  2. High-Resolution Solid Modeling of Biological Samples Imaged with 3D Fluorescence Microscopy

    PubMed Central

    Ferko, Michael C.; Patterson, Brian W.; Butler, Peter J.

    2011-01-01

    Optical-sectioning, digital fluorescence microscopy provides images representing temporally- and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling. PMID:16758474

  3. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    PubMed

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-03

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  4. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-01-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

  5. Developmental dynamics of IMSI-derived embryos: a time-lapse prospective study.

    PubMed

    Knez, Katja; Tomazevic, Tomaz; Vrtacnik-Bokal, Eda; Virant-Klun, Irma

    2013-08-01

    Because sperm vacuoles were marked as zones without chromatin in the sperm nucleus, which may reflect underlying chromosomal or DNA defects, this study considered whether they influence the morphology and dynamics of early developmental events in preimplantation embryos. Oocytes were injected with spermatozoa of four classes, according to the number and size of vacuoles at ×6000 magnification, and derived embryos were observed under time-lapse microscopy. For each embryo, the times of pronuclei appearance and disappearance and the first, second and third divisions were determined and related to its respective class of injected spermatozoa and its developmental stage. Embryos arising from normal class-I spermatozoa (without vacuoles) reached the 4-cell stage significantly earlier than embryos developed from class-IV spermatozoa (with large vacuoles and other abnormalities) (P=0.012). Blastocysts from class-I spermatozoa required the shortest mean time for all developmental events in comparison with blastocysts from spermatozoa of other classes (with vacuoles). Blastocysts also showed significantly earlier first division than arrested embryos in embryos arising from class-I spermatozoa (P=0.033). An insight into the developmental dynamics of embryo development according to morphology and head vacuoles of injected spermatozoa in morphologically selected sperm-derived embryos was observed for the first time.

  6. Modeling of time-lapse seismic reflection data from CO2 sequestration at West Pearl Queen Field

    NASA Astrophysics Data System (ADS)

    Bartel, L. C.; Haney, M. M.; Aldridge, D. F.; Symons, N. P.; Elbring, G. J.

    2006-12-01

    Sequestration of CO2 in depleted oil reservoirs, saline aquifers, or unminable coal sequences may prove to be an economical and environmentally safe means for long-term removal of carbon from the atmosphere. Requirements for storage of CO2 in subsurface geologic repositories (e.g., less than 0.1% per year leakage) pose significant challenges for geophysical remote sensing techniques. The many issues relevant to successful CO2 sequestration (volume in place, migration, leakage rate) require improved understanding of the advantages and pitfalls of potential monitoring methods. Advanced numerical modeling of time-lapse seismic reflection responses offers a controlled environment for testing hypotheses and exploring alternatives. The U.S. Department of Energy has conducted CO2 sequestration and monitoring tests at West Pearl Queen (WPQ) field in southeastern New Mexico. High-quality 9C/3D seismic reflection data were acquired before and after injection of ~2 kt of CO2 into a depleted sandstone unit at ~4200 ft depth. Images developed from time- lapse seismic data appear to reveal strong reflectivity changes attributed to displacement of brine by CO2. We are pursuing seismic numerical modeling studies with the goal of understanding and assessing the reliability and robustness of the time-lapse reflection responses. A 3D time-domain finite-difference isotropic elastic wave propagation algorithm generates realistic synthetic data. With this capability, we examine how various types of errors and noise in the 4D data degrade the ability to image a deep CO2 plume. Source/receiver sampling, subsurface illumination, correlated geologic heterogeneity, and static shifts are considered. As a result, we are able to make quantitative estimates of the tolerable errors for monitoring CO2 injection at WPQ field. Future plans include incorporating 3D poroelastic wave propagation modeling into the analysis. Sandia National Laboratories is a multiprogram science and engineering facility

  7. A high-throughput comparative characterization of laser-induced soft tissue damage using 3D digital microscopy.

    PubMed

    Das, Debobrato; Reed, Stephanie; Klokkevold, Perry R; Wu, Benjamin M

    2013-02-01

    3D digital microscopy was used to develop a rapid alternative approach to quantify the effects of specific laser parameters on soft tissue ablation and charring in vitro without the use of conventional tissue processing techniques. Two diode lasers operating at 810 and 980 nm wavelengths were used to ablate three tissue types (bovine liver, turkey breast, and bovine muscle) at varying laser power (0.3, 1.0, and 2.0 W) and velocities (1-50 mm/s). Spectrophotometric analyses were performed on each tissue to determine tissue-specific absorption coefficients and were considered in creating wavelength-dependent energy attenuation models to evaluate minimum heat of tissue ablations. 3D surface contour profiles characterizing tissue damage revealed that ablation depth and tissue charring increased with laser power and decreased with lateral velocity independent of wavelength and tissue type. While bovine liver ablation and charring were statistically higher at 810 than 980 nm (p < 0.05), turkey breast and bovine muscle ablated and charred more at 980 than 810 nm (p < 0.05). Spectrophotometric analysis revealed that bovine liver tissue had a greater tissue-specific absorption coefficient at 810 than 980 nm, while turkey breast and bovine muscle had a larger absorption coefficient at 980 nm (p < 0.05). This rapid 3D microscopic analysis of robot-driven laser ablation yielded highly reproducible data that supported well-defined trends related to laser-tissue interactions and enabled high throughput characterization of many laser-tissue permutations. Since 3D microscopy quantifies entire lesions without altering the tissue specimens, conventional and immunohistologic techniques can be used, if desired, to further interrogate specific sections of the digitized lesions.

  8. From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

    PubMed Central

    Tsai, Wen-Ting; Hassan, Ahmed; Sarkar, Purbasha; Correa, Joaquin; Metlagel, Zoltan; Jorgens, Danielle M.; Auer, Manfred

    2014-01-01

    Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data

  9. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

    DOE PAGES

    Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei; ...

    2015-08-17

    Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report that EMRinger is a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

  10. Image reconstruction for 3D light microscopy with a regularized linear method incorporating a smoothness prior

    NASA Astrophysics Data System (ADS)

    Preza, Chrysanthe; Miller, Michael I.; Conchello, Jose-Angel

    1993-07-01

    We have shown that the linear least-squares (LLS) estimate of the intensities of a 3-D object obtained from a set of optical sections is unstable due to the inversion of small and zero-valued eigenvalues of the point-spread function (PSF) operator. The LLS solution was regularized by constraining it to lie in a subspace spanned by the eigenvectors corresponding to a selected number of the largest eigenvalues. In this paper we extend the regularized LLS solution to a maximum a posteriori (MAP) solution induced by a prior formed from a 'Good's like' smoothness penalty. This approach also yields a regularized linear estimator which reduces noise as well as edge artifacts in the reconstruction. The advantage of the linear MAP (LMAP) estimate over the current regularized LLS (RLLS) is its ability to regularize the inverse problem by smoothly penalizing components in the image associated with small eigenvalues. Computer simulations were performed using a theoretical PSF and a simple phantom to compare the two regularization techniques. It is shown that the reconstructions using the smoothness prior, give superior variance and bias results compared to the RLLS reconstructions. Encouraging reconstructions obtained with the LMAP method from real microscopical images of a 10 micrometers fluorescent bead, and a four-cell Volvox embryo are shown.

  11. Web-based volume slicer for 3D electron-microscopy data from EMDB

    PubMed Central

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J.; Patwardhan, Ardan

    2016-01-01

    We describe the functionality and design of the Volume slicer – a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale. PMID:26876163

  12. 3D reconstruction and characterization of laser induced craters by in situ optical microscopy

    NASA Astrophysics Data System (ADS)

    Casal, A.; Cerrato, R.; Mateo, M. P.; Nicolas, G.

    2016-06-01

    A low-cost optical microscope was developed and coupled to an irradiation system in order to study the induced effects on material during a multipulse regime by an in situ visual inspection of the surface, in particular of the spot generated at different pulses. In the case of laser ablation, a reconstruction of the crater in 3D was made from the images of the sample surface taken during the irradiation process, and the subsequent profiles of ablated material were extracted. The implementation of this homemade optical device gives an added value to the irradiation system, providing information about morphology evolution of irradiated area when successive pulses are applied. In particular, the determination of ablation rates in real time can be especially useful for a better understanding and controlling of the ablation process in applications where removal of material is involved, such as laser cleaning and in-depth characterization of multilayered samples and diffusion processes. The validation of the developed microscope was made by a comparison with a commercial confocal microscope configured for the characterization of materials where similar results of crater depth and diameter were obtained for both systems.

  13. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy.

    PubMed

    Pawliczek, Piotr; Romanowska-Pawliczek, Anna; Soltys, Zbigniew

    2010-03-01

    Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson-Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines.

  14. A surface-based 3-D dendritic spine detection approach from confocal microscopy images.

    PubMed

    Li, Qing; Deng, Zhigang

    2012-03-01

    Determining the relationship between the dendritic spine morphology and its functional properties is a fundamental challenge in neurobiology research. In particular, how to accurately and automatically analyse meaningful structural information from a large microscopy image data set is far away from being resolved. As pointed out in existing literature, one remaining challenge in spine detection and segmentation is how to automatically separate touching spines. In this paper, based on various global and local geometric features of the dendrite structure, we propose a novel approach to detect and segment neuronal spines, in particular, a breaking-down and stitching-up algorithm to accurately separate touching spines. Extensive performance comparisons show that our approach is more accurate and robust than two state-of-the-art spine detection and segmentation algorithms.

  15. Time-lapse joint inversion of geophysical data with automatic joint constraints and dynamic attributes

    NASA Astrophysics Data System (ADS)

    Rittgers, J. B.; Revil, A.; Mooney, M. A.; Karaoulis, M.; Wodajo, L.; Hickey, C. J.

    2016-12-01

    Joint inversion and time-lapse inversion techniques of geophysical data are often implemented in an attempt to improve imaging of complex subsurface structures and dynamic processes by minimizing negative effects of random and uncorrelated spatial and temporal noise in the data. We focus on the structural cross-gradient (SCG) approach (enforcing recovered models to exhibit similar spatial structures) in combination with time-lapse inversion constraints applied to surface-based electrical resistivity and seismic traveltime refraction data. The combination of both techniques is justified by the underlying petrophysical models. We investigate the benefits and trade-offs of SCG and time-lapse constraints. Using a synthetic case study, we show that a combined joint time-lapse inversion approach provides an overall improvement in final recovered models. Additionally, we introduce a new approach to reweighting SCG constraints based on an iteratively updated normalized ratio of model sensitivity distributions at each time-step. We refer to the new technique as the Automatic Joint Constraints (AJC) approach. The relevance of the new joint time-lapse inversion process is demonstrated on the synthetic example. Then, these approaches are applied to real time-lapse monitoring field data collected during a quarter-scale earthen embankment induced-piping failure test. The use of time-lapse joint inversion is justified by the fact that a change of porosity drives concomitant changes in seismic velocities (through its effect on the bulk and shear moduli) and resistivities (through its influence upon the formation factor). Combined with the definition of attributes (i.e. specific characteristics) of the evolving target associated with piping, our approach allows localizing the position of the preferential flow path associated with internal erosion. This is not the case using other approaches.

  16. GPU-based rapid reconstruction of cellular 3D refractive index maps from tomographic phase microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dardikman, Gili; Shaked, Natan T.

    2016-03-01

    We present highly parallel and efficient algorithms for real-time reconstruction of the quantitative three-dimensional (3-D) refractive-index maps of biological cells without labeling, as obtained from the interferometric projections acquired by tomographic phase microscopy (TPM). The new algorithms are implemented on the graphic processing unit (GPU) of the computer using CUDA programming environment. The reconstruction process includes two main parts. First, we used parallel complex wave-front reconstruction of the TPM-based interferometric projections acquired at various angles. The complex wave front reconstructions are done on the GPU in parallel, while minimizing the calculation time of the Fourier transforms and phase unwrapping needed. Next, we implemented on the GPU in parallel the 3-D refractive index map retrieval using the TPM filtered-back projection algorithm. The incorporation of algorithms that are inherently parallel with a programming environment such as Nvidia's CUDA makes it possible to obtain real-time processing rate, and enables high-throughput platform for label-free, 3-D cell visualization and diagnosis.

  17. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton.

    PubMed

    Diaz-de-Quijano, Daniel; Palacios, Pilar; Horňák, Karel; Felip, Marisol

    2014-10-01

    The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), biases in individual 2D cell measurements were partially compensated, providing useful and comparable results to deconvolved 3D. We also discuss how much caution should be used when comparing the single-cell enzyme activities of different sized bacterio- and/or phytoplankton populations measured on 2D images. Finally, a novel method based on deconvolved 3D images (wide field restoration microscopy; WFR) was devised to improve the discrimination of similar single-cell enzyme activities, the comparison of enzyme activities between different size cells, the measurement of low fluorescence intensities, the quantification of less numerous species, and the combination of the FLEA technique with other single-cell methods. These improvements in cell enzyme activity measurements will provide a more precise picture of individual species' behavior in nature, which is essential to understand their functional role and evolutionary history.

  18. Measuring surface topography with scanning electron microscopy. I. EZEImage: a program to obtain 3D surface data.

    PubMed

    Ponz, Ezequiel; Ladaga, Juan Luis; Bonetto, Rita Dominga

    2006-04-01

    Scanning electron microscopy (SEM) is widely used in the science of materials and different parameters were developed to characterize the surface roughness. In a previous work, we studied the surface topography with fractal dimension at low scale and two parameters at high scale by using the variogram, that is, variance vs. step log-log graph, of a SEM image. Those studies were carried out with the FERImage program, previously developed by us. To verify the previously accepted hypothesis by working with only an image, it is indispensable to have reliable three-dimensional (3D) surface data. In this work, a new program (EZEImage) to characterize 3D surface topography in SEM has been developed. It uses fast cross correlation and dynamic programming to obtain reliable dense height maps in a few seconds which can be displayed as an image where each gray level represents a height value. This image can be used for the FERImage program or any other software to obtain surface topography characteristics. EZEImage also generates anaglyph images as well as characterizes 3D surface topography by means of a parameter set to describe amplitude properties and three functional indices for characterizing bearing and fluid properties.

  19. Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.

    PubMed

    Connell, Jodi L; Kim, Jiyeon; Shear, Jason B; Bard, Allen J; Whiteley, Marvin

    2014-12-23

    Microbes frequently live in nature as small, densely packed aggregates containing ∼10(1)-10(5) cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.

  20. 3-D Raman Imagery and Atomic Force Microscopy of Ancient Microscopic Fossils

    NASA Astrophysics Data System (ADS)

    Schopf, J.

    2003-12-01

    Investigations of the Precambrian (~540- to ~3,500-Ma-old) fossil record depend critically on identification of authentic microbial fossils. Combined with standard paleontologic studies (e.g., of paleoecologic setting, population structure, cellular morphology, preservational variants), two techniques recently introduced to such studies -- Raman imagery and atomic force microscopy -- can help meet this need. Laser-Raman imagery is a non-intrusive, non-destructive technique that can be used to demonstrate a micron-scale one-to-one correlation between optically discernable morphology and the organic (kerogenous) composition of individual microbial fossils(1,2), a prime indicator of biogencity. Such analyses can be used to characterize the molecular-structural makeup of organic-walled microscopic fossils both in acid-resistant residues and in petrographic thin sections, and whether the fossils analyzed are exposed at the upper surface of, or are embedded within (to depths >65 microns), the section studied. By providing means to map chemically, in three dimensions, whole fossils or parts of such fossils(3), Raman imagery can also show the presence of cell lumina, interior cellular cavities, another prime indicator of biogenicity. Atomic force microscopy (AFM) has been used to visualize the nanometer-scale structure of the kerogenous components of single Precambrian microscopic fossils(4). Capable of analyzing minute fragments of ancient organic matter exposed at the upper surface of thin sections (or of kerogen particles deposited on flat surfaces), such analyses hold promise not only for discriminating between biotic and abiotic micro-objects but for elucidation of the domain size -- and, thus, the degree of graphitization -- of the graphene subunits of the carbonaceous matter analyzed. These techniques -- both new to paleobiology -- can provide useful insight into the biogenicity and geochemical maturity of ancient organic matter. References: (1) Kudryavtsev, A.B. et

  1. Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

    PubMed Central

    Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

    2016-01-01

    Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

  2. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.

    PubMed

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup; Flo, Trude Helen; Halaas, Øyvind

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.

  3. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

    PubMed Central

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. PMID:26406896

  4. Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy.

    PubMed

    King, Sharon V; Doblas, Ana; Patwary, Nurmohammed; Saavedra, Genaro; Martínez-Corral, Manuel; Preza, Chrysanthe

    2015-10-10

    Spatial light modulator (SLM) implementation of wavefront encoding enables various types of engineered point-spread functions (PSFs), including the generalized-cubic and squared-cubic phase mask wavefront encoded (WFE) PSFs, shown to reduce the impact of sample-induced spherical aberration in fluorescence microscopy. This investigation validates dynamic experimental parameter variation of these WFE-PSFs. We find that particular design parameter bounds exist, within which the divergence of computed and experimental WFE-PSFs is of the same order of magnitude as that of computed and experimental conventional PSFs, such that model-based approaches for solving the inverse imaging problem can be applied to a wide range of SLM-WFE systems. Interferometric measurements were obtained to evaluate the SLM implementation of the numeric mask. Agreement between experiment and theory in terms of a wrapped phase, 0-2π, validates the phase mask implementation and allows characterization of the SLM response. These measurements substantiate experimental practice of computational-optical microscope imaging with an SLM-engineered PSF.

  5. High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

    PubMed Central

    Braun, Alexander; Caesar, Nicole M.; Dang, Kyvan; Myers, Kenneth A.

    2016-01-01

    The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration. PMID:27584860

  6. 3D image reconstruction using optical sectioning in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Seo, Jungwoo; Kang, Dong Kyun; Park, Sunglim; Gweon, Dae gab

    2001-10-01

    Confocal scanning microscopy (CSM) has been used in biological application, materials science, semiconductor quality measurement and other non-destructive microscopic application. Small spot of light illuminates a sample, and a small detector that is ideally a point detector collects the reflected or transmitted light having the information of specimen. An image distribution can be reconstructed by a correlation analysis of spots with the high bandwidth. The mechanism for two-dimensional beam scanning and optical sectioning has an important role in CSM as the three-dimensional profiler. The parasitic motion of focus on the detector gives rise to the fatal distortion of an image profile named the extinction effect while using acousto-optical (AO) deflector. The intensity profile for the open loop scanning should be matched with its response for the standard. The non-linearity can be minimized with the optical sectioning or the optical probe of the closed loop control. This paper shows the mathematical expression of the light such as the extinction curve in the optical fields of system using AO deflector, the axial/lateral response experimentally when the error sources change, and the methods of optical sectioning. We propose the progressive methods for the high quality image as the following. At first, for having the corrected image, small spot and long scan range, this paper shows that the optimal design having the multi-objects can be used by choosing the unitary lens device in CSM. At second, in order to compensate for the intensity cancellation at the end profile that may be the cause of waviness for the optical image, this paper shows that it is efficient to schedule the frequency of scan. According to characteristics of the extinction curve and axial/lateral response having the error property, we can define the frequency and sensitivity of as their robustness. Finally, the axial response gives an important motive for the optical section, and the limit of

  7. Source Repeatability of Time-Lapse Offset VSP Surveys for Monitoring CO2 Injection

    NASA Astrophysics Data System (ADS)

    Zhang, Z.; Huang, L.; Rutledge, J. T.; Denli, H.; Zhang, H.; McPherson, B. J.; Grigg, R.

    2009-12-01

    Time-lapse vertical seismic profiling (VSP) surveys have the potential to remotely track the migration of injected CO2 within a geologic formation. To accurately detect small changes due to CO2 injection, the sources of time-lapse VSP surveys must be located exactly at the same positions. However, in practice, the source locations can vary from one survey to another survey. Our numerical simulations demonstrate that a variation of a few meters in the VSP source locations can result in significant changes in time-lapse seismograms. To address the source repeatability issue, we apply double-difference tomography to downgoing waves of time-lapse offset VSP data to invert for the source locations and the velocity structures simultaneously. In collaboration with Resolute Natural Resources, Navajo National Oil and Gas Company, and the Southwest Regional Partnership on Carbon Sequestration under the support of the U.S. Department of Energy’s National Energy Technology Laboratory, one baseline and two repeat offset VSP datasets were acquired in 2007-2009 for monitoring CO2 injection at the Aneth oil field in Utah. A cemented geophone string was used to acquire the data for one zero-offset and seven offset source locations. During the data acquisition, there was some uncertainty in the repeatability of the source locations relative to the baseline survey. Our double-difference tomography results of the Aneth time-lapse VSP data show that the source locations for different surveys are separated up to a few meters. Accounting for these source location variations during VSP data analysis will improve reliability of time-lapse VSP monitoring.

  8. Advances in interpretation of subsurface processes with time-lapse electrical imaging

    SciTech Connect

    Singha, Kamini; Day-Lewis, Frederick D.; Johnson, Timothy C.; Slater, Lee D.

    2015-03-15

    Electrical geophysical methods, including electrical resistivity, time-domain induced polarization, and complex resistivity, have become commonly used to image the near subsurface. Here, we outline their utility for time-lapse imaging of hydrological, geochemical, and biogeochemical processes, focusing on new instrumentation, processing, and analysis techniques specific to monitoring. We review data collection procedures, parameters measured, and petrophysical relationships and then outline the state of the science with respect to inversion methodologies, including coupled inversion. We conclude by highlighting recent research focused on innovative applications of time-lapse imaging in hydrology, biology, ecology, and geochemistry, among other areas of interest.

  9. Time-lapse in the IVF-lab: how should we assess potential benefit?

    PubMed

    Armstrong, S; Vail, A; Mastenbroek, S; Jordan, V; Farquhar, C

    2015-01-01

    Time-lapse imaging of embryos has been widely introduced to fertility laboratories worldwide with the aim of identifying the best quality embryos to transfer that will ultimately improve IVF success rates. In this opinion paper, we explore the lack of evidence of benefit of this novel intervention, analyse the methodological flaws of current studies, offer ideal study designs that assess the various features of time-lapse imaging, and discuss forthcoming studies. In particular, we emphasize the ethical aspects of hastily adopting a costly technology without current high level evidence of improved live birth rates, safety and cost effectiveness.

  10. Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture

    PubMed Central

    Johnson, Thomas V.; Oglesby, Ericka N.; Steinhart, Matthew R.; Cone-Kimball, Elizabeth; Jefferys, Joan; Quigley, Harry A.

    2016-01-01

    Purpose To develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments. Methods Thy1-YFP mouse retinas were explanted and maintained in organotypic culture. Retinal ganglion cell dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing. Results Retinal ganglion cells in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra- and interobserver measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently > 0.80). Statistically significant (P < 0.001) dendritic tree degeneration was detected following 7 days in culture including: 40% to 50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF+CNTF significantly attenuated dendritic field degeneration. Conclusions Retinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning within individual RGCs. PMID:26811145

  11. Microfluidics-integrated time-lapse imaging for analysis of cellular dynamics.

    PubMed

    Albrecht, Dirk R; Underhill, Gregory H; Resnikoff, Joshua; Mendelson, Avital; Bhatia, Sangeeta N; Shah, Jagesh V

    2010-06-01

    An understanding of the mechanisms regulating cellular responses has recently been augmented by innovations enabling the observation of phenotypes at high spatio-temporal resolution. Technologies such as microfluidics have sought to expand the throughput of these methods, although assimilation with advanced imaging strategies has been limited. Here, we describe the pairing of high resolution time-lapse imaging with microfluidic multiplexing for the analysis of cellular dynamics, utilizing a design selected for facile fabrication and operation, and integration with microscopy instrumentation. This modular, medium-throughput platform enables the long-term imaging of living cells at high numerical aperture (via oil immersion) by using a conserved 96-well, approximately 6 x 5 mm(2) imaging area with a variable input/output channel design chosen for the number of cell types and microenvironments under investigation. In the validation of this system, we examined fundamental features of cell cycle progression, including mitotic kinetics and spindle orientation dynamics, through the high-resolution parallel analysis of model cell lines subjected to anti-mitotic agents. We additionally explored the self-renewal kinetics of mouse embryonic stem cells, and demonstrate the ability to dynamically assess and manipulate stem cell proliferation, detect rare cell events, and measure extended time-scale correlations. We achieved an experimental throughput of >900 cells/experiment, each observed at >40x magnification for up to 120 h. Overall, these studies illustrate the capacity to probe cellular functions and yield dynamic information in time and space through the integration of a simple, modular, microfluidics-based imaging platform.

  12. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    PubMed Central

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-01-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

  13. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    PubMed

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  14. High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

    PubMed Central

    Holden, Seamus J.; Pengo, Thomas; Meibom, Karin L.; Fernandez Fernandez, Carmen; Collier, Justine; Manley, Suliana

    2014-01-01

    We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of “Z-ring” organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization. PMID:24616530

  15. Holographic microscopy and microfluidics platform for measuring wall stress and 3D flow over surfaces textured by micro-pillars

    PubMed Central

    Bocanegra Evans, Humberto; Gorumlu, Serdar; Aksak, Burak; Castillo, Luciano; Sheng, Jian

    2016-01-01

    Understanding how fluid flow interacts with micro-textured surfaces is crucial for a broad range of key biological processes and engineering applications including particle dispersion, pathogenic infections, and drag manipulation by surface topology. We use high-speed digital holographic microscopy (DHM) in combination with a correlation based de-noising algorithm to overcome the optical interference generated by surface roughness and to capture a large number of 3D particle trajectories in a microfluidic channel with one surface patterned with micropillars. It allows us to obtain a 3D ensembled velocity field with an uncertainty of 0.06% and 2D wall shear stress distribution at the resolution of ~65 μPa. Contrary to laminar flow in most microfluidics, we find that the flow is three-dimensional and complex for the textured microchannel. While the micropillars affect the velocity flow field locally, their presence is felt globally in terms of wall shear stresses at the channel walls. These findings imply that micro-scale mixing and wall stress sensing/manipulation can be achieved through hydro-dynamically smooth but topologically rough micropillars. PMID:27353632

  16. In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-05-01

    Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

  17. Multiphoton microscopy of engineered dermal substitutes: assessment of 3D collagen matrix remodeling induced by fibroblasts contraction

    NASA Astrophysics Data System (ADS)

    Pena, A.-M.; Olive, C.; Michelet, J.-F.; Galey, J.-B.; Fagot, D.; Leroy, F.; Martin, J.-L.; Colonna, A.; Schanne-Klein, M.-C.

    2010-02-01

    One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction.

  18. Volumetric label-free imaging and 3D reconstruction of mammalian cochlea based on two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Xianzeng; Geng, Yang; Ye, Qing; Zhan, Zhenlin; Xie, Shusen

    2013-11-01

    The visualization of the delicate structure and spatial relationship of intracochlear sensory cells has relied on the laborious procedures of tissue excision, fixation, sectioning and staining for light and electron microscopy. Confocal microscopy is advantageous for its high resolution and deep penetration depth, yet disadvantageous due to the necessity of exogenous labeling. In this study, we present the volumetric imaging of rat cochlea without exogenous dyes using a near-infrared femtosecond laser as the excitation mechanism and endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. We find that TPEF exhibits strong contrast, allowing cellular and even subcellular resolution imaging of the cochlea, differentiating cell types, visualizing delicate structures and the radial nerve fiber. Our results further demonstrate that 3D reconstruction rendered with z-stacks of optical sections enables better revealment of fine structures and spatial relationships, and easily performed morphometric analysis. The TPEF-based optical biopsy technique provides great potential for new and sensitive diagnostic tools for hearing loss or hearing disorders, especially when combined with fiber-based microendoscopy.

  19. A 3D High Frequency Array Based 16 Channel Photoacoustic Microscopy System for In Vivo Micro-vascular Imaging

    PubMed Central

    Zemp, Roger; Yen, Jesse; Wang, L.V.; Shung, K. Kirk

    2009-01-01

    This paper discusses the design of a novel photoacoustic microscopy imaging system with promise for studying the structure of tissue microvasculature for applications in visualizing angiogenesis. A new sixteen channel analog and digital high frequency array based photoacoustic microscopy system (PAM) was developed using an Nd:YLF pumped tunable dye laser, a 30MHz piezo composite linear array transducer and a custom multi-channel receiver electronics system. Using offline delay and sum beamforming and beamsteering, phantom images were obtained from a 6µm carbon fiber in water at a depth of 8mm. The measured -6dB lateral and axial spatial resolution of the system was 100±5µm and 45±5µm, respectively. The dynamic focusing capability of the system was demonstrated by imaging a composite carbon fiber matrix through a 12.5mm imaging depth. Next, 2-D in vivo images were formed of vessels around 100µm in diameter in the human hand. 3-D in vivo images were also formed of micro-vessels 3mm below the surface of the skin in two Sprague Dawley rats. PMID:19131292

  20. Time-Lapse Motion Picture Technique Applied to the Study of Geological Processes.

    PubMed

    Miller, R D; Crandell, D R

    1959-09-25

    Light-weight, battery-operated timers were built and coupled to 16-mm motion-picture cameras having apertures controlled by photoelectric cells. The cameras were placed adjacent to Emmons Glacier on Mount Rainier. The film obtained confirms the view that exterior time-lapse photography can be applied to the study of slow-acting geologic processes.

  1. Feasibility of monitoring gas hydrate production with time-lapse VSP

    SciTech Connect

    Kowalsky, M.B.; Nakagawa, S.; Moridis, G.J.

    2009-11-01

    In this work we begin to examine the feasibility of using time-lapse seismic methods-specifically the vertical seismic profiling (VSP) method-for monitoring changes in hydrate accumulations that are predicted to occur during production of natural gas.

  2. Estimating topsoil water content of clay soils with data from time-lapse electrical conductivity surveys

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spatial estimation of soil water content (') at the field, hillslope, or catchment scale is required in numerous applications. Time-lapse electrical resistivity and electrical conductivity surveys were recognized as the useful source of information about both spatial variations in soil water conten...

  3. 2D Time-lapse Seismic Tomography Using An Active Time Constraint (ATC) Approach

    EPA Science Inventory

    We propose a 2D seismic time-lapse inversion approach to image the evolution of seismic velocities over time and space. The forward modeling is based on solving the eikonal equation using a second-order fast marching method. The wave-paths are represented by Fresnel volumes rathe...

  4. First successful pregnancies following embryo selection using Time-lapse technology in Iran: Case report

    PubMed Central

    Faramarzi, Azita; Khalili, Mohammad Ali; Soleimani, Mehrdad

    2015-01-01

    Background: Embryo selection is a vital part of in vitro fertilization (IVF) programs, with morphology-based grading systems having been widely used for decades. Time-lapse imaging combined with embryo morph kinetics may proffer a non-invasive means for improving embryo selection. We report the first ongoing and chemical pregnancies using Time-lapse embryo scope to select best embryos for transfer in Iran. Cases: A case with tubal factor infertility was admitted to IVF program with normozoospermia. After ovarian hyper stimulation, 6 COCs were retrieved and inseminated with 25,000 progressive sperms/ oocyte. Five zygotes were placed individually into the micro wells of equilibrated embryo scope dish for Time-lapse observation, and incubated at 37°C, 5% CO2. On day 3, single embryo transfer (SET) took place based on kinetic parameters of the embryos. Clinical pregnancy was confirmed 7 weeks after SET. The second case with history of previous ICSI failure was admitted with azoospermia. Nine MII oocytes underwent ICSI, and incubated in Time-lapse facilities. The rest of procedures were followed as described for case 1. Chemical pregnancy was confirmed 15 days after SET. Conclusion: This approach opens a way to select best embryo non-invasively for SET; thus, increasing implantation, while reducing multiple pregnancy complications. PMID:26131014

  5. Time-lapse motion picture technique applied to the study of geological processes

    USGS Publications Warehouse

    Miller, R.D.; Crandell, D.R.

    1959-01-01

    Light-weight, battery-operated timers were built and coupled to 16-mm motion-picture cameras having apertures controlled by photoelectric cells. The cameras were placed adjacent to Emmons Glacier on Mount Rainier. The film obtained confirms the view that exterior time-lapse photography can be applied to the study of slow-acting geologic processes.

  6. Full waveform inversion of repeating seismic events to estimate time-lapse velocity changes

    NASA Astrophysics Data System (ADS)

    Kamei, R.; Lumley, D.

    2017-02-01

    Seismic monitoring provides valuable information regarding the time-varying changes in subsurface physical properties caused by natural or man-made processes. However, the resulting changes in the earth's subsurface properties are often small both in terms of magnitude and spatial extent, leading to minimal time-lapse differences in seismic amplitude or traveltime. In order to better extract information from the time-lapse data, we show that exploiting the full seismic waveform information can be critical. In this study, we develop and test methods of full waveform inversion that estimate an optimal subsurface model of time-varying elastic properties in order to fit the observed time-lapse seismic data with predicted waveforms based on numerical solutions of the wave equation. Time-lapse full waveform inversion is non-linear and non-unique, and depends on the knowledge of the baseline velocity model before a change, and (non-)repeatability of earthquake source and sensor parameters, and of ambient and cultural noise. We propose to use repeating earthquake data sets acquired with permanent arrays of seismic sensors to enhance the repeatability of source and sensor parameters. We further develop and test time-lapse parallel, double-difference and bootstrapping inversion strategies to mitigate the dependence on the baseline velocity model. The parallel approach uses a time-invariant full waveform inversion method to estimate velocity models independently of the different source event times. The double-difference approach directly estimates velocity changes from time-lapse waveform differences, requiring excellent repeatability. The bootstrapping approach inverts for velocity models sequentially in time, implicitly constraining the time-lapse inversions, while relaxing an explicit requirement for high data repeatability. We assume that prior to the time-lapse inversion, we can estimate the true source locations and the origin time of the events, and also we can also

  7. Structure-Function Studies of Blood and Air Capillaries in Chicken Lung Using 3D Electron Microscopy

    PubMed Central

    West, John B.; Fu, Zhenxing; Deerinck, Thomas J.; Mackey, Mason R.; Obayashi, James T.; Ellisman, Mark H.

    2010-01-01

    Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure. A possible mechanism for these differences is that avian pulmonary capillaries, unlike mammalian, are supported from the outside by air capillaries, but the details of the support are poorly understood. To clarify this we studied the blood and air capillaries in chicken lung using transmission electron microscopy (EM) and two relatively new techniques that allow 3D visualization: electron tomography and serial block-face scanning EM. These studies show that the pulmonary capillaries are flanked by epithelial bridges composed of two extremely thin epithelial cells with large surface areas. The junctions of the bridges with the capillary walls show thickening of the epithelial cells and an accumulation of extracellular matrix. Collapse of the pulmonary capillaries when the pressure outside them is increased is apparently prevented by the guy wire-like action of the epithelial bridges. The enlarged junctions between the bridges and the walls could provide a mechanism that limits the hoop stress in the capillary walls when the pressure inside them is increased. The support of the pulmonary capillaries may also be explained by an interdependence mechanism whereby the capillaries are linked to a rigid assemblage of air capillaries. These EM studies show the supporting structures in greater detail than has previously been possible, particularly in 3D, and they allow a more complete analysis of the mechanical forces affecting avian pulmonary capillaries. PMID:20038456

  8. Using Digital Time-Lapse Videos to Teach Geomorphic Processes to Undergraduates

    NASA Astrophysics Data System (ADS)

    Clark, D. H.; Linneman, S. R.; Fuller, J.

    2004-12-01

    We demonstrate the use of relatively low-cost, computer-based digital imagery to create time-lapse videos of two distinct geomorphic processes in order to help students grasp the significance of the rates, styles, and temporal dependence of geologic phenomena. Student interviews indicate that such videos help them to understand the relationship between processes and landform development. Time-lapse videos have been used extensively in some sciences (e.g., biology - http://sbcf.iu.edu/goodpract/hangarter.html, meteorology - http://www.apple.com/education/hed/aua0101s/meteor/, chemistry - http://www.chem.yorku.ca/profs/hempsted/chemed/home.html) to demonstrate gradual processes that are difficult for many students to visualize. Most geologic processes are slower still, and are consequently even more difficult for students to grasp, yet time-lapse videos are rarely used in earth science classrooms. The advent of inexpensive web-cams and computers provides a new means to explore the temporal dimension of earth surface processes. To test the use of time-lapse videos in geoscience education, we are developing time-lapse movies that record the evolution of two landforms: a stream-table delta and a large, natural, active landslide. The former involves well-known processes in a controlled, repeatable laboratory experiment, whereas the latter tracks the developing dynamics of an otherwise poorly understood slope failure. The stream-table delta is small and grows in ca. 2 days; we capture a frame on an overhead web-cam every 3 minutes. Before seeing the video, students are asked to hypothesize how the delta will grow through time. The final time-lapse video, ca. 20-80 MB, elegantly shows channel migration, progradation rates, and formation of major geomorphic elements (topset, foreset, bottomset beds). The web-cam can also be "zoomed-in" to show smaller-scale processes, such as bedload transfer, and foreset slumping. Post-lab tests and interviews with students indicate that

  9. Time-lapse monitoring of localized changes within heterogeneous media with scattered waves

    NASA Astrophysics Data System (ADS)

    Chinaemerem, Kanu

    Time-lapse monitoring of geological and mechanical media has been the focus of various studies over the past four decades because of the information that the inferred changes within the medium provides insight into the dynamic characteristics of the medium. Time-lapse changes within a medium can be used to characterize the temporal evolution of the medium, evaluate the forces driving the changes within the medium and make predictions on the future state of the monitored medium. The detectability of the changes within a material depends on the characteristics of the change to be imaged, the sensitivity of the monitoring data to the change, and the time-lapse monitoring parameters such as the monitoring source-receiver array and the spectral content of the monitoring waves. Various time-lapse monitoring tools have been used to monitor changes within media ranging from the earth's surface to tumors within the human body. These monitoring tools include the use of 4D active surveys were an imprint of the change within the medium is extracted from the time-lapse surveys and the use of interferometric techniques that use singly or multiply scattered waves. My major goal in this study is to image and localize changes present within a scattering medium using time-lapse multiply scattered waves generated within the monitored medium. The changes to be imaged are generally localized in space. This work is an extension of coda wave interferometry. Coda wave interferometry focuses on the identification and extraction of average velocity change occurring within a scattering medium. Due to the non-linear characteristics of multiply scattered waves and limited information of the origin of the multiply scattered waves, coda wave interferometry resolves the average velocity change within the scattering medium with no or limited indication of the location of the change. In this study, I demonstrate that time-lapse changes can be imaged and localized within scattering media using

  10. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

    Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

    2016-08-01

    Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

  11. A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage

    PubMed Central

    Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

    2016-01-01

    The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

  12. Correction of Depth-Dependent Aberrations in 3D Single Molecule Localization and Super-resolution Microscopy

    PubMed Central

    McGorty, Ryan; Schnitzbauer, Joerg; Zhang, Wei; Huang, Bo

    2014-01-01

    Single molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive-index-mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here, we show a method to obtain accurate, depth dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane. PMID:24562125

  13. Optical sectioning and 3D reconstructions as an alternative to scanning electron microscopy for analysis of cell shape1

    PubMed Central

    Landis, Jacob B.; Ventura, Kayla L.; Soltis, Douglas E.; Soltis, Pamela S.; Oppenheimer, David G.

    2015-01-01

    Premise of the study: Visualizing flower epidermal cells is often desirable for investigating the interaction between flowers and their pollinators, in addition to the broader range of ecological interactions in which flowers are involved. We developed a protocol for visualizing petal epidermal cells without the limitations of the commonly used method of scanning electron microscopy (SEM). Methods: Flower material was collected and fixed in glutaraldehyde, followed by dehydration in an ethanol series. Flowers were dissected to collect petals, and subjected to a Histo-Clear series to remove the cuticle. Material was then stained with aniline blue, mounted on microscope slides, and imaged using a compound fluorescence microscope to obtain optical sections that were reconstructed into a 3D image. Results: This optical sectioning method yielded high-quality images of the petal epidermal cells with virtually no damage to cells. Flowers were processed in larger batches than are possible using common SEM methods. Also, flower size was not a limiting factor as often observed in SEM studies. Flowers up to 5 cm in length were processed and mounted for visualization. Conclusions: This method requires no special equipment for sample preparation prior to imaging and should be seen as an alternative method to SEM. PMID:25909040

  14. Time-Lapse 3D Inversion of Complex Conductivity Data Using an Active Time Constrained (ATC) Approach

    EPA Science Inventory

    Induced polarization (more precisely the magnitude and the phase of the impedance of the subsurface) is measured using a network of electrodes located at the ground surface or in boreholes. This method yields important information related to the distribution of permeability and ...

  15. Monitoring CO 2 sequestration into deep saline aquifer and associated salt intrusion using coupled multiphase flow modeling and time lapse electrical resistivity tomography

    SciTech Connect

    Chuan Lu; CHI Zhang; Hai Hanag; Timothy C. Johnson

    2014-04-01

    Successful geological storage and sequestration of carbon dioxide (CO2) require efficient monitoring of the migration of CO2 plume during and after large-scale injection in order to verify the containment of the injected CO2 within the target formation and to evaluate potential leakage risk. Field studies have shown that surface and cross-borehole electrical resistivity tomography (ERT) can be a useful tool in imaging and characterizing solute transport in heterogeneous subsurface. In this synthetic study, we have coupled a 3-D multiphase flow model with a parallel 3-D time-lapse ERT inversion code to explore the feasibility of using time-lapse ERT for simultaneously monitoring the migration of CO2 plume in deep saline formation and potential brine intrusion into shallow fresh water aquifer. Direct comparisons of the inverted CO2 plumes resulting from ERT with multiphase flow simulation results indicate the ERT could be used to delineate the migration of CO2 plume. Detailed comparisons on the locations, sizes and shapes of CO2 plume and intruded brine plumes suggest that ERT inversion tends to underestimate the area review of the CO2 plume, but overestimate the thickness and total volume of the CO2 plume. The total volume of intruded brine plumes is overestimated as well. However, all discrepancies remain within reasonable ranges. Our study suggests that time-lapse ERT is a useful monitoring tool in characterizing the movement of injected CO2 into deep saline aquifer and detecting potential brine intrusion under large-scale field injection conditions.

  16. Integrating Terrestrial Time-Lapse Photography with Laser Scanning to Distinguish the Drivers of Movement at Sólheimajökull, Iceland

    NASA Astrophysics Data System (ADS)

    How, P.; James, M. R.; Wynn, P.

    2014-12-01

    Glacier movement is attributed to a sensitive configuration of driving forces. Here, we present an approach designed to evaluate the drivers of movement at Sólheimajökull, an outlet glacier from the Myrdalsjökull ice cap, Iceland, through combining terrestrial time-lapse photography and laser scanning (TLS). A time-lapse camera (a dSLR with intervalometer and solar-recharged battery power supply) collected hourly data over the summer of 2013. The data are subject to all the difficulties that are usually present in long time-lapse sequences, such as highly variable illumination and visibility conditions, evolving surfaces, and camera instabilities. Feature-tracking software [1] was used to: 1) track regions of static topography (e.g. the skyline) from which camera alignment could be continuously updated throughout the sequence; and 2) track glacial surface features for velocity estimation. Absolute georeferencing of the image sequence was carried out by registering the camera to a TLS survey acquired at the beginning of the monitoring period. A second TLS survey (July 2013) provided an additional 3D surface. By assuming glacial features moved in approximately planimetrically straight lines between the two survey dates, combining the two TLS surfaces with the monoscopic feature tracking allows 3D feature tracks to be derived. Such tracks will enable contributions from different drivers (e.g. surface melting) to be extracted, even in imagery that is acquired not perpendicular to glacier motion. At Sólheimajökull, our aim is to elucidate any volcanic contribution to the observed movement.[1] http://www.lancaster.ac.uk/staff/jamesm/software/pointcatcher.htm

  17. Freeze core sampling to validate time-lapse resistivity monitoring of the hyporheic zone.

    PubMed

    Toran, Laura; Hughes, Brian; Nyquist, Jonathan; Ryan, Robert

    2013-01-01

    A freeze core sampler was used to characterize hyporheic zone storage during a stream tracer test. The pore water from the frozen core showed tracer lingered in the hyporheic zone after the tracer had returned to background concentration in collocated well samples. These results confirmed evidence of lingering subsurface tracer seen in time-lapse electrical resistivity tomographs. The pore water exhibited brine exclusion (ion concentrations in ice lower than source water) in a sediment matrix, despite the fast freezing time. Although freeze core sampling provided qualitative evidence of lingering tracer, it proved difficult to quantify tracer concentration because the amount of brine exclusion during freezing could not be accurately determined. Nonetheless, the additional evidence for lingering tracer supports using time-lapse resistivity to detect regions of low fluid mobility within the hyporheic zone that can act as chemically reactive zones of importance in stream health.

  18. Calixarenes and cations: a time-lapse photography of the big-bang.

    PubMed

    Casnati, Alessandro

    2013-08-07

    The outstanding cation complexation properties emerging from the pioneering studies on calixarene ligands during a five-year period in the early 1980s triggered a big-bang burst of publications on such macrocycles that is still lasting at a distance of more than 30 years. A time-lapse photography of this timeframe is proposed which allows the readers to pinpoint the contributions of the different research groups.

  19. Imaging Hydrological Processes in Headwater Riparian Seeps with Time-Lapse Electrical Resistivity.

    PubMed

    Williams, Mark R; Buda, Anthony R; Singha, Kamini; Folmar, Gordon J; Elliott, Herschel A; Schmidt, John P

    2017-01-01

    Delineating hydrologic and pedogenic factors influencing groundwater flow in riparian zones is central in understanding pathways of water and nutrient transport. In this study, we combined two-dimensional time-lapse electrical resistivity imaging (ERI) (depth of investigation approximately 2 m) with hydrometric monitoring to examine hydrological processes in the riparian area of FD-36, a small (0.4 km(2) ) agricultural headwater basin in the Valley and Ridge region of east-central Pennsylvania. We selected two contrasting study sites, including a seep with groundwater discharge and an adjacent area lacking such seepage. Both sites were underlain by a fragipan at 0.6 m. We then monitored changes in electrical resistivity, shallow groundwater, and nitrate-N concentrations as a series of storms transitioned the landscape from dry to wet conditions. Time-lapse ERI revealed different resistivity patterns between seep and non-seep areas during the study period. Notably, the seep displayed strong resistivity reductions (∼60%) along a vertically aligned region of the soil profile, which coincided with strong upward hydraulic gradients recorded in a grid of nested piezometers (0.2- and 0.6-m depth). These patterns suggested a hydraulic connection between the seep and the nitrate-rich shallow groundwater system below the fragipan, which enabled groundwater and associated nitrate-N to discharge through the fragipan to the surface. In contrast, time-lapse ERI indicated no such connections in the non-seep area, with infiltrated rainwater presumably perched above the fragipan. Results highlight the value of pairing time-lapse ERI with hydrometric and water quality monitoring to illuminate possible groundwater and nutrient flow pathways to seeps in headwater riparian areas.

  20. Time-lapse AVO fluid inversion for dynamic reservoir characterization in Delhi Field, Louisiana

    NASA Astrophysics Data System (ADS)

    Putri, Indah Hermansyah

    In the development stage, CO2 injection is becoming more widely used in enhanced oil recovery (EOR). Delhi Oil Field is part of Phases XIII and XIV of the Reservoir Characterization Project (RCP) Colorado School of Mines. The focus of these phases is to monitor the effectiveness of the CO 2 injection in Delhi Field by using multicomponent time-lapse seismic data. In this study, I analyze the amplitude versus offset (AVO) response of the time-lapse P-wave seismic data in order to quantify the fluid probability in the field. RCP acquired four square miles of multicomponent time-lapse seismic in Delhi Field to characterize the field dynamically. RCP's two surveys, monitor 1 and monitor 2, were shot in 2010 and 2011 after the start of CO2 injection in November 2009. Time-lapse AVO modeling was performed. The modeling results show that both the top Tuscaloosa and Paluxy Formations are class III AVO, and change toward class IV AVO by increasing the CO2 saturation in the reservoir. In addition, the Paluxy Formation shows a consistent result between the synthetic and real data, however, the Tuscaloosa Formation is not consistent as it is affected by tuning. AVO fluid inversion (AFI) was performed on both the Tuscaloosa and Paluxy Formations in order to quantify the fluid probability in these formations. The inversion results are confirmed by the pseudo gamma ray model, the porosity model, the permeability model, the pressure model, and the production data. In the Tuscaloosa and Paluxy Formations, oil and CO2 are located in the good quality, high porosity, and high permeability sandstones. The presence of CO2 is also confirmed by the pressure interpretation. Furthermore, production data from both Tuscaloosa and Paluxy Formations confirm the fluid presence in the reservoir.

  1. Time-lapse cross-hole electrical resistivity tomography monitoring effects of an urban tunnel

    NASA Astrophysics Data System (ADS)

    Bellmunt, F.; Marcuello, A.; Ledo, J.; Queralt, P.; Falgàs, E.; Benjumea, B.; Velasco, V.; Vázquez-Suñé, E.

    2012-12-01

    Tunnel construction in urban areas has recently become a topic of interest and has increased the use of tunnel boring machines. Monitoring subsurface effects due to tunnel building in urban areas with conventional surface geophysical techniques is not an easy task because of space constraints. Taking advantage of the construction of a new metro line in Barcelona (Spain), a geoelectrical experiment, which included borehole logging and time-lapse cross-hole measurements using permanent electrode deployments, was designed to characterise and to study the subsurface effects of the tunnel drilling in a test site. We present a case study in which the differences between time-lapse cross-hole resistivity measurements acquired before, during and after the tunnel drilling below the test site have been calculated using three different procedures: a constrained time-lapse inversion, a model subtraction and an inversion of the normalised data ratio. The three procedures have provided satisfactory images of the resistivity changes and tunnel geometry, but resistivity changes for the tunnel void were lower than predicted by modelling. This behaviour has been explained by considering a conductive zone around the tunnel. Further, an apparent resistivity pseudosection for the cross-hole data, equivalent to the case of the equatorial dipole-dipole on the surface, is introduced.

  2. Efficiency of time-lapse intervals and simple baits for camera surveys of wild pigs

    USGS Publications Warehouse

    Williams, B.L.; Holtfreter, R.W.; Ditchkoff, S.S.; Grand, J.B.

    2011-01-01

    Growing concerns surrounding established and expanding populations of wild pigs (Sus scrofa) have created the need for rapid and accurate surveys of these populations. We conducted surveys of a portion of the wild pig population on Fort Benning, Georgia, to determine if a longer time-lapse interval than had been previously used in surveys of wild pigs would generate similar detection results. We concurrently examined whether use of soured corn at camera sites affected the time necessary for pigs to locate a new camera site or the time pigs remained at a site. Our results suggest that a 9-min time-lapse interval generated dependable detection results for pigs and that soured corn neither attracted pigs to a site any quicker than plain, dry, whole-kernel corn, nor held them at a site longer. Maximization of time-lapse interval should decrease data and processing loads, and use of a simple, available bait should decrease cost and effort associated with more complicated baits; combination of these concepts should increase efficiency of wild pig surveys. ?? 2011 The Wildlife Society.

  3. Estimating Flow Properties from the Onset of Time-Lapse Changes

    NASA Astrophysics Data System (ADS)

    Vasco, D. W.

    2015-12-01

    Geophysical time-lapse observations are increasingly important for monitoring subsurface fluid flow. Time-lapse data can even be used to characterize spatial variations in the properties of a porous medium. A major impediment to such characterization is the difficulty in connecting flow-related changes to changes geophysical properties. For example, physical models used to relate changes in fluid saturation and pressure to seismic velocity changes often depend upon unknown parameters, or on the detailed distribution of the fluid at intermediate spatial scales. The challange is particularly acute when one tries to relate the magnitudes of saturation and pressure changes to the magnitude of a change in a time-lapse observation. I present an alternative approach for the characterization of a porous medium, based upon the onset of changes of a geophysical attribute, that is applicable when there are a sequence of geophysical surveys. An onset time is the calander time as which a geophysical observable begins to deviate from its initial or background value. In many cases onset times are sensitive to flow properties, such as permeability, and insensitive to the details of the physical model governing the geophysical response. Several examples of the use of onset times will be presented, including the seismic monitoring of injected carbon dioxide and the use of surface deformation data to image fluid flow at depth. Though the technique works best when there are numerous geophysical snapshots, numerical modeling indicates useful results are possible from yearly seismic surveys.

  4. Using time-lapse photogrammetric method to study the terminal part of the Perito Moreno glacier

    NASA Astrophysics Data System (ADS)

    Lenzano, M. G.; Toth, C.; Lenzano, L.; Skvarca, P.; Smalley, R.

    2013-05-01

    The changes that are occurring in regional climate affect cryospheric environments and have a direct impact on the hydrological cycle. This work presents a feasibility study on the implementation and performance assessment of time-lapse processing ofstereo image sequence, acquired by calibrated cameras, in order to determine the altimetric and volumetric changes in the terminus of Perito Moreno (PM) glacier. This glacier is located at 50° 28' 23''S, 73° 02' 10''W at the Parque Nacional Los Glaciares, South Patagonia Icefield, Santa Cruz, Argentina. This glacier has experienced minor fluctuations or unusual behavior with respect to others glaciers since early 1960's until nowadays. The time-lapse technique allows for obtaining accurate estimates of deformations and velocity models. Digital Terrain Models (DTMs) were created from images, captured daily from April, 2012 to November, 2012, with a total of 182 days. One of the challenges was maintaining the accurate co-registration of the DTMs, which was essential for the information extraction. The differences between DTMs provided the velocities in the terminal part of PM for the period as it approaches the Peninsula de Magallanes. In additon, the DTMs were validated in order to determine the degree of uncertainty in the estimation of changes in the glacier. Keywords: time-lapse, DTMs, glacier volume change, Perito Moreno.

  5. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

  6. Gabor-domain optical coherence microscopy with integrated dual-axis MEMS scanner for fast 3D imaging and metrology

    NASA Astrophysics Data System (ADS)

    Canavesi, Cristina; Cogliati, Andrea; Hayes, Adam; Santhanam, Anand P.; Tankam, Patrice; Rolland, Jannick P.

    2015-10-01

    Fast, robust, nondestructive 3D imaging is needed for characterization of microscopic structures in industrial and clinical applications. A custom micro-electromechanical system (MEMS)-based 2D scanner system was developed to achieve 55 kHz A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) instrument with a novel multilevel GPU architecture for high-speed imaging. GD-OCM yields high-definition volumetric imaging with dynamic depth of focusing through a bio-inspired liquid lens-based microscope design, which has no moving parts and is suitable for use in a manufacturing setting or in a medical environment. A dual-axis MEMS mirror was chosen to replace two single-axis galvanometer mirrors; as a result, the astigmatism caused by the mismatch between the optical pupil and the scanning location was eliminated and a 12x reduction in volume of the scanning system was achieved. Imaging at an invariant resolution of 2 μm was demonstrated throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. The MEMS-based scanner resulted in improved image quality, increased robustness and lighter weight of the system - all factors that are critical for on-field deployment. A custom integrated feedback system consisting of a laser diode and a position-sensing detector was developed to investigate the impact of the resonant frequency of the MEMS and the driving signal of the scanner on the movement of the mirror. Results on the metrology of manufactured materials and characterization of tissue samples with GD-OCM are presented.

  7. Embryonic lineage analysis using three-dimensional, time-lapse in-vivo fluorescent microscopy

    NASA Astrophysics Data System (ADS)

    Minden, Jonathan; Kam, Zvi; Agard, David A.; Sedat, John W.; Alberts, Bruce

    1990-08-01

    Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.

  8. Enhancing Monitoring of Recharge-Related Environmental Remediation Processes Using Time-Lapse Seismic Refraction

    NASA Astrophysics Data System (ADS)

    Gaines, D. P.; Baker, G. S.; Hubbard, S. S.; Watson, D. B.; Jardine, P. M.

    2008-12-01

    The application of time-lapse seismic methods has typically been constrained to large-scale geologic investigations associated with petroleum exploration and exploitation; however, there is growing interest in monitoring near-surface phenomena (e.g., fluid flow in fractured or karstic geologic media, hydraulic recharge, and near-surface anthropogenic manipulations) using time-lapse seismic methods. In order to demonstrate the feasibility of detailed time-lapse seismic refraction tomography (TLSRT), we have monitored a perched water table at Oak Ridge National Laboratory (ORNL) Y-12 site in conjunction with a multi- disciplinary investigation of the fate and transport of contaminants. Due to remnant anthropogenic alterations of the site (i.e., replacement of 0-7 meters of contaminated soil with poorly sorted limestone gravel fill during construction of a seepage basin cap), the near surface hydrology is extremely complex and is hypothesized to have a large influence on infiltration, contaminant distribution, and contaminant remobilization. Understanding the impact of recharge-related flow and transport processes is especially important in regions that are subjected to significant precipitation events, such as at the ORNL Y-12 site. Here, TLSRT techniques are used to monitor the changing geometry of a perched water table located near the covered seepage basin, while coincident time-lapse surface electrical resistivity (TLERT) measurements are used to monitor changes in total dissolved solids due to recharge-related dilution. Data are collected at multiple time intervals (i.e., daily, weekly, monthly, yearly) and at varying stages in the evolution of the perch zone. The resulting seismic data are processed using wavepath eikonal tomography (WET) and differenced to identify areas of variable velocity associated with a change in saturation. The differenced tomograms correlate with discrete point water table measurements; however, the highly variable water table at this

  9. The application of time-lapse photography for the observation of snow processes in mountainous catchments

    NASA Astrophysics Data System (ADS)

    Garvelmann, J.; Pohl, S.; Weiler, M.

    2012-04-01

    For the forecast of snowmelt flood events in mountainous catchments it is very important to know the spatial distribution and temporal evolution of the snowcover. Topography and vegetation have the most important influence on the spatio-temporal variability of the snowcover. In order to accomplish a continuous observation of the quantity and the status of the snowcover, an extensive measurement network consisting of numerous standalone snow and meteorological sensors and time-lapse photography was established in three catchments in the Black Forest, a typical mid latitude medium elevation mountain range. Catchments with different topographic characteristic and areal extent were specifically chosen for this study. Within the catchments, a stratified sampling design was used to cover a wide range of altitudes and exposures. In order to investigate the influence of a vegetation cover on the snow processes beneath sensors and cameras have been installed under the forest canopy and on adjacent open field sites, respectively. In the presented study the application of spatially distributed time-lapse cameras for the observation of snow processes and snowcover properties at the catchment scale will be discussed. Image analysis software was applied to extract information about snowdepth, snow albedo and canopy interception from the digital images. A measurement scale with a black/white board was installed in the focus of every camera to allow a determination of the snowdepth at every camera location while the black/white board was used to provide a white balance for the albedo estimation. The albedo provides important information about the status of the snowcover and its temporal evolution is a crucial factor for the snowmelt energy balance. Furthermore the time-lapse images provided a continuous observation of the forest canopy allowing the estimation of the interception efficiency and the temporal evolution of the snow interception for different topographic situations

  10. Rapid, simple and inexpensive production of custom 3D printed equipment for large-volume fluorescence microscopy

    PubMed Central

    Tyson, Adam L.; Hilton, Stephen T.; Andreae, Laura C.

    2015-01-01

    The cost of 3D printing has reduced dramatically over the last few years and is now within reach of many scientific laboratories. This work presents an example of how 3D printing can be applied to the development of custom laboratory equipment that is specifically adapted for use with the novel brain tissue clearing technique, CLARITY. A simple, freely available online software tool was used, along with consumer-grade equipment, to produce a brain slicing chamber and a combined antibody staining and imaging chamber. Using standard 3D printers we were able to produce research-grade parts in an iterative manner at a fraction of the cost of commercial equipment. 3D printing provides a reproducible, flexible, simple and cost-effective method for researchers to produce the equipment needed to quickly adopt new methods. PMID:25797056

  11. Application of time-lapse ERT to Characterize Soil-Water-Disease Interactions of Citrus Orchard - Case Study

    NASA Astrophysics Data System (ADS)

    Peddinti, S. R.; Kbvn, D. P.; Ranjan, S.; Suradhaniwar, S.; J, P. A.; R M, G.

    2015-12-01

    Vidarbha region in Maharashtra, India (home for mandarin Orange) experience severe climatic uncertainties resulting in crop failure. Phytopthora are the soil-borne fungal species that accumulate in the presence of moisture, and attack the root / trunk system of Orange trees at any stage. A scientific understanding of soil-moisture-disease relations within the active root zone under different climatic, irrigation, and crop cycle conditions can help in practicing management activities for improved crop yield. In this study, we developed a protocol for performing 3-D time-lapse electrical resistivity tomography (ERT) at micro scale resolution to monitor the changes in resistivity distribution within the root zone of Orange trees. A total of 40 electrodes, forming a grid of 3.5 m x 2 m around each Orange tree were used in ERT survey with gradient and Wenner configurations. A laboratory test on un-disturbed soil samples of the region was performed to plot the variation of electrical conductivity with saturation. Curve fitting techniques were applied to get the modified Archie's model parameters. The calibrated model was further applied to generate the 3-D soil moisture profiles of the study area. The point estimates of soil moisture were validated using TDR probe measurements at 3 different depths (10, 20, and 40 cm) near to the root zone. In order to understand the effect of soil-water relations on plant-disease relations, we performed ERT analysis at two locations, one at healthy and other at Phytopthora affected Orange tree during the crop cycle, under dry and irrigated conditions. The degree to which an Orange tree is affected by Phytopthora under each condition is evaluated using 'grading scale' approach following visual inspection of the canopy features. Spatial-temporal distribution of moisture profiles is co-related with grading scales to comment on the effect of climatic and irrigation scenarios on the degree and intensity of crop disease caused by Phytopthora.

  12. High-Resolution Time-Lapse Monitoring of Unsaturated Flow using Automated GPR Data Collection

    NASA Astrophysics Data System (ADS)

    Mangel, A. R.; Moysey, S. M.; Lytle, B. A.; Bradford, J. H.

    2015-12-01

    High-resolution ground-penetrating radar (GPR) data provide the detailed information required to image subsurface structures. Recent advances in GPR monitoring now also make it possible to study transient hydrologic processes, but high-speed data acquisition is critical for this application. We therefore highlight the capabilities of our automated system to acquire time-lapse, high-resolution multifold GPR data during infiltration of water into soils. The system design allows for fast acquisition of constant-offset (COP) and common-midpoint profiles (CMP) to monitor unsaturated flow at multiple locations. Qualitative interpretation of the unprocessed COPs can provide substantial information regarding the hydrologic response of the system, such as the complexities of patterns associated with the wetting of the soil and geophysical evidence of non-uniform propagation of a wetting front. While we find that unprocessed images are informative, we show that the spatial variability of velocity introduced by infiltration events can complicate the images and that migration of the data is an effective tool to improve interpretability of the time-lapse images. The ability of the system to collect high density CMP data also introduces the potential for improving the velocity model along with the image via reflection tomography in the post-migrated domain. We show that for both simulated and empirical time-lapse GPR profiles we can resolve a propagating wetting front in the soil that is in good agreement with the response of in-situ soil moisture measurements. The data from these experiments illustrate the importance of high-speed, high-resolution GPR data acquisition for obtaining insight about the dynamics of hydrologic events. Continuing research is aimed at improving the quantitative analysis of surface-based GPR monitoring data for identifying preferential flow in soils.

  13. Time-Lapse Electrical Geophysical Monitoring of Amendment-Based Biostimulation.

    PubMed

    Johnson, Timothy C; Versteeg, Roelof J; Day-Lewis, Frederick D; Major, William; Lane, John W

    2015-01-01

    Biostimulation is increasingly used to accelerate microbial remediation of recalcitrant groundwater contaminants. Effective application of biostimulation requires successful emplacement of amendment in the contaminant target zone. Verification of remediation performance requires postemplacement assessment and contaminant monitoring. Sampling-based approaches are expensive and provide low-density spatial and temporal information. Time-lapse electrical resistivity tomography (ERT) is an effective geophysical method for determining temporal changes in subsurface electrical conductivity. Because remedial amendments and biostimulation-related biogeochemical processes often change subsurface electrical conductivity, ERT can complement and enhance sampling-based approaches for assessing emplacement and monitoring biostimulation-based remediation. Field studies demonstrating the ability of time-lapse ERT to monitor amendment emplacement and behavior were performed during a biostimulation remediation effort conducted at the Department of Defense Reutilization and Marketing Office (DRMO) Yard, in Brandywine, Maryland, United States. Geochemical fluid sampling was used to calibrate a petrophysical relation in order to predict groundwater indicators of amendment distribution. The petrophysical relations were field validated by comparing predictions to sequestered fluid sample results, thus demonstrating the potential of electrical geophysics for quantitative assessment of amendment-related geochemical properties. Crosshole radar zero-offset profile and borehole geophysical logging were also performed to augment the data set and validate interpretation. In addition to delineating amendment transport in the first 10 months after emplacement, the time-lapse ERT results show later changes in bulk electrical properties interpreted as mineral precipitation. Results support the use of more cost-effective surface-based ERT in conjunction with limited field sampling to improve spatial

  14. Exploring the Resolution of Time-Lapse Microgravity at an Aquifer Storage and Recovery Site

    NASA Astrophysics Data System (ADS)

    Bradley, C. C.; Ali, M.; Levannier, A.

    2008-12-01

    Time-Lapse Microgravity can reveal relatively small underground fluid displacements via the redistribution of density-contrast at fluid boundaries. The method has, for example, been successfully demonstrated in underground natural gas storage and carbon sequestration studies, and has clear potential for hydrological research and applications such as mining and hydrocarbon reservoir management. For surveys based on modern spring-type gravimeters, the technique's basic measurement resolution is limited by instrument drift, offset errors, and viscoelastic strain hysteresis of the sensor. Despite the sophistication of modern instruments, actual field performance of gravimeters still depends on operator technique and survey conditions. To explore the ultimate resolution of field microgravity (and time-lapse measurements, in particular), we have made repeated gravity surveys of a shallow aquifer storage and recovery test site in the UAE, where injection produces a 'water mound' - localized vertical water-level shifts - monitored by a set of instrumented wells. Based on field measurements and additional laboratory testing of our Scintrex CG-5 gravimeter, we find that the main limit on measurement resolution is from orientation strain hysteresis - a variable amplitude error that decays in approximately 30 minutes, typically in response to orientation during transport between measurement stations. Still, carefully conducted surveys (during the summer, in the desert conditions of the UAE) demonstrate time-lapse microgravity resolution of about 3 microGals, corresponding to a water-level shift of about 0.3 m for this aquifer. In this paper, we will discuss what a 'careful survey' requires and present our detailed testing and survey results.

  15. Time-Lapse Electrical Geophysical Monitoring of Amendment-Based Biostimulation

    SciTech Connect

    Johnson, Timothy C.; Versteeg, Roelof; Day-Lewis, Frederick D.; Major, William; Lane, John W.

    2015-12-02

    Biostimulation is increasingly used to accelerate microbial remediation of recalcitrant groundwater contaminants. Effective application of biostimulation requires successful emplacement of amendment in the contaminant target zone. Verification of remediation performance requires postemplacement assessment and contaminant monitoring. Sampling based approaches are expensive and provide low-density spatial and temporal information. Time-lapse electrical resistivity tomography (ERT) is an effective geophysical method for determining temporal changes in subsurface electrical conductivity. Because remedial amendments and biostimulation-related biogeochemical processes often change subsurface electrical conductivity, ERT can complement and enhance sampling-based approaches for assessing emplacement and monitoring biostimulation-based remediation. Field studies demonstrating the ability of time-lapse ERT to monitor amendment emplacement and behavior were performed during a biostimulation remediation effort conducted at the Department of Defense Reutilization and Marketing Office (DRMO) Yard, in Brandywine, Maryland, United States. Geochemical fluid sampling was used to calibrate a petrophysical relation in order to predict groundwater indicators of amendment distribution. The petrophysical relations were field validated by comparing predictions to sequestered fluid sample results, thus demonstrating the potential of electrical geophysics for quantitative assessment of amendment-related geochemical properties. Crosshole radar zero-offset profile and borehole geophysical logging were also performed to augment the data set and validate interpretation. In addition to delineating amendment transport in the first 10 months after emplacement, the time-lapse ERT results show later changes in bulk electrical properties interpreted as mineral precipitation. Results support the use of more cost-effective surfacebased ERT in conjunction with limited field sampling to improve spatial

  16. A state-space Bayesian framework for estimating biogeochemical transformations using time-lapse geophysical data

    SciTech Connect

    Chen, J.; Hubbard, S.; Williams, K.; Pride, S.; Li, L.; Steefel, C.; Slater, L.

    2009-04-15

    We develop a state-space Bayesian framework to combine time-lapse geophysical data with other types of information for quantitative estimation of biogeochemical parameters during bioremediation. We consider characteristics of end-products of biogeochemical transformations as state vectors, which evolve under constraints of local environments through evolution equations, and consider time-lapse geophysical data as available observations, which could be linked to the state vectors through petrophysical models. We estimate the state vectors and their associated unknown parameters over time using Markov chain Monte Carlo sampling methods. To demonstrate the use of the state-space approach, we apply it to complex resistivity data collected during laboratory column biostimulation experiments that were poised to precipitate iron and zinc sulfides during sulfate reduction. We develop a petrophysical model based on sphere-shaped cells to link the sulfide precipitate properties to the time-lapse geophysical attributes and estimate volume fraction of the sulfide precipitates, fraction of the dispersed, sulfide-encrusted cells, mean radius of the aggregated clusters, and permeability over the course of the experiments. Results of the case study suggest that the developed state-space approach permits the use of geophysical datasets for providing quantitative estimates of end-product characteristics and hydrological feedbacks associated with biogeochemical transformations. Although tested here on laboratory column experiment datasets, the developed framework provides the foundation needed for quantitative field-scale estimation of biogeochemical parameters over space and time using direct, but often sparse wellbore data with indirect, but more spatially extensive geophysical datasets.

  17. A state-space Bayesian framework for estimating biogeochemical transformations using time-lapse geophysical data

    NASA Astrophysics Data System (ADS)

    Chen, Jinsong; Hubbard, Susan S.; Williams, Kenneth H.; Pride, Steve; Li, Li; Steefel, Carl; Slater, Lee

    2009-08-01

    We develop a state-space Bayesian framework to combine time-lapse geophysical data with other types of information for quantitative estimation of biogeochemical parameters during bioremediation. We consider characteristics of end products of biogeochemical transformations as state vectors, which evolve under constraints of local environments through evolution equations, and consider time-lapse geophysical data as available observations, which could be linked to the state vectors through petrophysical models. We estimate the state vectors and their associated unknown parameters over time using Markov chain Monte Carlo sampling methods. To demonstrate the use of the state-space approach, we apply it to complex resistivity data collected during laboratory column biostimulation experiments that were poised to precipitate iron and zinc sulfides during sulfate reduction. We develop a petrophysical model based on sphere-shaped cells to link the sulfide precipitate properties to the time-lapse geophysical attributes and estimate volume fraction of the sulfide precipitates, fraction of the dispersed, sulfide-encrusted cells, mean radius of the aggregated clusters, and permeability over the course of the experiments. Results of the case study suggest that the developed state-space approach permits the use of geophysical data sets for providing quantitative estimates of end-product characteristics and hydrological feedbacks associated with biogeochemical transformations. Although tested here on laboratory column experiment data sets, the developed framework provides the foundation needed for quantitative field-scale estimation of biogeochemical parameters over space and time using direct, but often sparse wellbore data with indirect, but more spatially extensive geophysical data sets.

  18. Time-lapse camera studies of sea-disposed chemical munitions in Hawaii

    NASA Astrophysics Data System (ADS)

    Edwards, Margo H.; Fornari, Daniel J.; Rognstad, Mark R.; Kelley, Christopher D.; Mah, Christopher L.; Davis, Logan K.; Flores, Kyle R. M.; Main, Erin L.; Bruso, Natalie L.

    2016-06-01

    The interactions between fauna and sea-disposed munitions provide important evidence regarding whether munitions constituents affect the health of the ocean environment and its inhabitants. To date few studies of these interactions have been conducted at deep-water disposal sites; typically observations of fauna in the vicinity of sea-disposed munitions are limited to the few minutes or hours required to collect physical samples at a specific location. During the 2012 Hawaii Undersea Military Munitions Assessment (HUMMA) field program we deployed two deep-sea time-lapse camera systems with the objectives of cataloging the diversity of fauna visiting sea-disposed chemical munitions and observing faunal behavior and physiology. Over the 1- and 3-day deployments we recorded 28 different species of fishes, crustaceans, mollusks, cnidarians, and echinoderms at the two sites. Both cameras captured the previously undocumented behavior of brisingid sea stars repositioning themselves along chemical munitions casings. Despite the fact that brisingid sea stars are able to move, for the duration of both time-lapse experiments they remained on chemical munitions casings. We interpret this result to indicate that the advantages of residing on a hard substrate slightly elevated above the seafloor outweigh the effects of chemical munitions constituents for brisingid sea stars. One type of physiological anomaly observed on several arms of the brisingid sea stars at the time-lapse sites led to the collection and examination of six specimens. As reported by Mah (2015. Deep Sea Res. II, 2015, XX-XX), these physiological features are the result of parasitic crustaceans and are not caused by chemical munitions constituents.

  19. Time-Lapse Imaging to Examine the Growth Kinetics of Arabidopsis Seedlings in Response to Ethylene.

    PubMed

    Binder, Brad M

    2017-01-01

    Ethylene is well known to inhibit the growth of dark-grown eudicot seedlings. Most studies examine this inhibition after several days of exposure to ethylene. However, such end-point analysis misses transient responses and the dynamic nature of growth regulation. Here, high-resolution, time-lapse imaging is described as a method to gather data about ethylene growth kinetics and movement responses of the hypocotyls of dark-grown seedlings of Arabidopsis thaliana. These methods allow for the characterization of short-term kinetic responses and can be modified for the analysis of roots and seedlings from other species.

  20. Arctic sea-ice variations from time-lapse passive microwave imagery

    NASA Technical Reports Server (NTRS)

    Campbell, W. J.; Ramseier, R. O.; Zwally, H. J.; Gloersen, P.

    1980-01-01

    This paper presents: (1) a short historical review of the passive microwave research on sea ice, which established the observational and theoretical base permitting the interpretation of the first passive microwave images of earth obtained by the Nimbus-5 ESMR; (2) the construction of a time-lapse motion picture film of a 16-month set of serial ESMR images to aid in the formidable data analysis task; and (3) a few of the most significant findings resulting from an early analysis of these data, using selected ESMR images to illustrate these findings.

  1. Time-lapse and slow-motion tracking of temperature changes: response time of a thermometer

    NASA Astrophysics Data System (ADS)

    Moggio, L.; Onorato, P.; Gratton, L. M.; Oss, S.

    2017-03-01

    We propose the use of a smartphone based time-lapse and slow-motion video techniques together with tracking analysis as valuable tools for investigating thermal processes such as the response time of a thermometer. The two simple experimental activities presented here, suitable also for high school and undergraduate students, allow one to measure in a simple yet rigorous way the response time of an alcohol thermometer and show its critical dependence on the properties of the surrounding environment giving insight into instrument characteristics, heat transfer and thermal equilibrium concepts.

  2. In vitro ovarian tumor growth and treatment response dynamics visualized with time-lapse OCT imaging

    PubMed Central

    Evans, Conor L.; Rizvi, Imran; Hasan, Tayyaba; de Boer, Johannes F.

    2010-01-01

    In vitro three-dimensional models for metastatic ovarian cancer have been useful for recapitulating the human disease. These spheroidal tumor cultures, however, can grow in excess of 1 mm in diameter, which are difficult to visualize without suitable imaging technology. Optical coherence tomography (OCT) is an ideal live imaging method for non-perturbatively visualizing these complex systems. OCT enabled detailed observations of the model at both nodular and cellular levels, revealing growth dynamics not previously observed. The development of a time-lapse OCT system, capable of automated, multidimensional acquisition, further provided insights into the growth and chemotherapeutic response of ovarian cancer. PMID:19466138

  3. Arctic sea-ice variations from time-lapse passive microwave imagery

    USGS Publications Warehouse

    Campbell, W.J.; Ramseier, R.O.; Zwally, H.J.; Gloersen, P.

    1980-01-01

    This paper presents: (1) a short historical review of the passive microwave research on sea ice which established the observational and theoretical base permitting the interpretation of the first passive microwave images of Earth obtained by the Nimbus-5 ESMR; (2) the construction of a time-lapse motion picture film of a 16-month set of serial ESMR images to aid in the formidable data analysis task; and (3) a few of the most significant findings resulting from an early analysis of these data, using selected ESMR images to illustrate these findings. ?? 1980 D. Reidel Publishing Co.

  4. Live-streaming: Time-lapse video evidence of novel streamer formation mechanism and varying viscosity

    PubMed Central

    Parvinzadeh Gashti, Mazeyar; Bellavance, Julien; Kroukamp, Otini; Wolfaardt, Gideon; Taghavi, Seyed Mohammad; Greener, Jesse

    2015-01-01

    Time-lapse videos of growing biofilms were analyzed using a background subtraction method, which removed camouflaging effects from the heterogeneous field of view to reveal evidence of streamer formation from optically dense biofilm segments. In addition, quantitative measurements of biofilm velocity and optical density, combined with mathematical modeling, demonstrated that streamer formation occurred from mature, high-viscosity biofilms. We propose a streamer formation mechanism by sudden partial detachment, as opposed to continuous elongation as observed in other microfluidic studies. Additionally, streamer formation occurred in straight microchannels, as opposed to serpentine or pseudo-porous channels, as previously reported. PMID:26339304

  5. Live-streaming: Time-lapse video evidence of novel streamer formation mechanism and varying viscosity.

    PubMed

    Parvinzadeh Gashti, Mazeyar; Bellavance, Julien; Kroukamp, Otini; Wolfaardt, Gideon; Taghavi, Seyed Mohammad; Greener, Jesse

    2015-07-01

    Time-lapse videos of growing biofilms were analyzed using a background subtraction method, which removed camouflaging effects from the heterogeneous field of view to reveal evidence of streamer formation from optically dense biofilm segments. In addition, quantitative measurements of biofilm velocity and optical density, combined with mathematical modeling, demonstrated that streamer formation occurred from mature, high-viscosity biofilms. We propose a streamer formation mechanism by sudden partial detachment, as opposed to continuous elongation as observed in other microfluidic studies. Additionally, streamer formation occurred in straight microchannels, as opposed to serpentine or pseudo-porous channels, as previously reported.

  6. Soil Contamination, Advanced integrated characterisation and time-lapse Monitoring, SoilCAM project highlights

    NASA Astrophysics Data System (ADS)

    French, H. K.; Van Der Zee, S. E.; Wehrer, M.; Godio, A.; Pedersen, L. B.; Tsocano, G.

    2013-12-01

    The SoilCAM project (2008- 2012, EU-FP7-212663) aimed at improving methods for monitoring subsurace contaminant distribution and biodegradation. Two test sites were chosen, Oslo airport Gardermoen, Norway where de-icing agents infiltrate the soil during snowmelt and the Trecate site in Italy where an inland crude oil spill occurred in 1994. A number of geophysical investigation techniques were combined with soil and water sampling techniques. Data obtained from time-lapse measurements were further analysed by numerical modelling of flow and transport at different scales in order to characterise transport processes in the unsaturated and saturated zones. Laboratory experiments provided physical and biogeochemical data for model parameterisation and to select remediation methods. The geophysical techniques were used to map geological heterogeneities and to conduct time-lapse measurements of processes in the unsaturated zone. Both cross borehole and surface electrodes were used for electrical resistivity and induced polarisation surveys. Results showed clear indications of areas highly affected by de-icing chemicals along the runway at Oslo airport. The time lapse measurements along the runway at the airport showed infiltration patterns during snowmelt and were used to validate 2D unsaturated flow and transport simulations using SUTRA. The simulations illustrate the effect of layering geological structures and membranes, buried parallel to the runway, on the flow pattern. Complex interaction between bio-geo-chemical processes in a 1D vertical profile along the runway were described with the ORCHESTRA model. Smaller scale field site measurements revealed increase of iron and manganese during degradation of de-icing chemicals. At the Trecate site a combination of georadar, electrical resistivity and radio magneto telluric provided a broad outline of the geology down to 50 m. Anomalies in the Induced polarisation and electrical resistivity data from the cross borehole

  7. A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

    PubMed Central

    Kirkby, Paul A.; Naga Srinivas, N.K.M.; Silver, R. Angus

    2010-01-01

    We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 μm; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space PMID:20588506

  8. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy

    PubMed Central

    Rupprecht, Peter; Prendergast, Andrew; Wyart, Claire; Friedrich, Rainer W

    2016-01-01

    There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio. PMID:27231612

  9. Preliminary Seismic Time-lapse Results of the First Post-injection Monitoring at the Ketzin Pilot Site for CO2 Storage

    NASA Astrophysics Data System (ADS)

    Huang, Fei; Monika, Ivandic; Julin, Christopher; Lüth, Stefan; Bergmann, Peter

    2016-04-01

    Time-lapse surface seismic monitoring methods have proven to be notably successful in imaging the development of the CO2 plume injected into the 630-650 meters deep saline aquifer at the Ketzin pilot site. A 3D baseline survey was acquired in the autumn of 2005 prior to CO2 injection, followed by two 3D repeat surveys conducted in the same season of 2009 and 2012 after injection of about 22 and 61 kt of CO2, respectively. These repeat surveys showed that the CO2 plume was concentrated around the injection well with a preferred WNW propagating trend due to reservoir heterogeneity. The CO2 induced amplitude anomaly continued to grow with more CO2 injected into the reservoir. The CO2 injection ended on August 29, 2013 after a total of about 67 kt injected CO2. In the autumn of 2015, a third 3D repeat survey with a nominal fold of 25 was acquired during the post-injection phase. The acquisition was implemented with an identical template scheme and acquisition parameters as in the previous surveys. About 5500 source points were acquired during the 57 days of active acquisition. The same processing steps as used before were carried out from pre-stack to post-stack. In order to minimize changes unrelated to the site operations, cross-calibration was applied to the data. Preliminary results of the time-lapse analysis show that the obtained amplitude anomaly at the reservoir level is smaller in size than the one observed at the time of the second repeat survey. This may indicate that CO2 dissolution is active, especially east of the injection site, where permeability and flow rate appear to be lower. In comparison to the previous surveys, the maximum amplitude anomaly has moved towards the west, which is consistent with the previously observed propagating tendency of the CO2 plume within the reservoir. No CO2 leakage is observed within the overburden.

  10. The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization.

    PubMed

    Scalco, Eleonora; Amato, Alberto; Ferrante, Maria Immacolata; Montresor, Marina

    2016-11-01

    Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.

  11. Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

    PubMed Central

    Zopf, Christopher J.; Maheshri, Narendra

    2013-01-01

    Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications. PMID:23892428

  12. Retinal flat cells participate in the formation of fibers by retinal neuroblasts in vitro. Time lapse video studies.

    PubMed

    Li, H P; Sheffield, J B

    1986-03-01

    Freshly dissociated cells from embryonic chick neural retinas grow in characteristic patterns on flat cells or on chick embryo mesodermal cells. A striking difference between the two patterns is that the cells grown on flat cells are interconnected by a complex network of fibers, whereas those grown on mesodermal cells are aggregated into clusters that remain relatively isolated within the mesodermal monolayer. Analysis by time-lapse video microscopy indicates that two processes produce the fibers. (1) Fibers grow out by the extension of growth cones from cells within aggregates. (2) Neuronal cell aggregates that attach to two flat cells are pulled apart by the movement of the cells beneath them. As the aggregate is pulled apart, portions of the cells remain attached to the two halves, and their cytoplasm is drawn into thin fibers. The lack of fibers on a mesodermal substrate is due to two factors: (1) Aggregates are widely spaced on the substrate surface and do not come into contact often. (2) On those occasions when they do come into contact, the movement of the monolayer is so vigorous that emerging fibers are torn.

  13. Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

    NASA Astrophysics Data System (ADS)

    Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

    2014-02-01

    Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

  14. Time lapse seismic observations and effects of reservoir compressibility at Teal South oil field

    NASA Astrophysics Data System (ADS)

    Islam, Nayyer

    One of the original ocean-bottom time-lapse seismic studies was performed at the Teal South oil field in the Gulf of Mexico during the late 1990's. This work reexamines some aspects of previous work using modern analysis techniques to provide improved quantitative interpretations. Using three-dimensional volume visualization of legacy data and the two phases of post-production time-lapse data, I provide additional insight into the fluid migration pathways and the pressure communication between different reservoirs, separated by faults. This work supports a conclusion from previous studies that production from one reservoir caused regional pressure decline that in turn resulted in liberation of gas from multiple surrounding unproduced reservoirs. I also provide an explanation for unusual time-lapse changes in amplitude-versus-offset (AVO) data related to the compaction of the producing reservoir which, in turn, changed an isotropic medium to an anisotropic medium. In the first part of this work, I examine regional changes in seismic response due to the production of oil and gas from one reservoir. The previous studies primarily used two post-production ocean-bottom surveys (Phase I and Phase II), and not the legacy streamer data, due to the unavailability of legacy prestack data and very different acquisition parameters. In order to incorporate the legacy data in the present study, all three post-stack data sets were cross-equalized and examined using instantaneous amplitude and energy volumes. This approach appears quite effective and helps to suppress changes unrelated to production while emphasizing those large-amplitude changes that are related to production in this noisy (by current standards) suite of data. I examine the multiple data sets first by using the instantaneous amplitude and energy attributes, and then also examine specific apparent time-lapse changes through direct comparisons of seismic traces. In so doing, I identify time-delays that, when

  15. Numerical studies of imaging subsurface waterfloods using CSERT with time-lapse inversion

    NASA Astrophysics Data System (ADS)

    Zhang, Ying-Ying; Liu, De-Jun; Wang, Li-Yang; Ai, Qing-Hui; Qin, Min-Jun

    2015-12-01

    During the operation of the waterflooding technique, it is necessary to identify the waterflooding areas to enhance oil displacement efficiency. A casing-surface electrical resistivity tomography (CSERT) system using a well casing as a long electrode is able to detect a wide lateral scope, but its vertical resolution and ability to identify deep anomalies in the reservoir are limited, particularly for reservoirs with high-conductivity anomalies in the shallow subsurface, which disturb the response from the water floods at depth. In this study, we first simplified this kind of reservoir into a dual layered anomaly model. Then the log-inject-log method with time-lapse inversion was proposed and evaluated regarding its ability to improve the imaging of the deep waterflooding areas and the shallow anomaly. The results were compared with the commonly used static measurement with static inversion. In the static inversion results, the shallow anomaly was imaged well but the deep anomaly was unobservable. The results of the proposed log-inject-log method with time-lapse inversion showed that it is able to identify the shallow and deep anomalies better under various conditions, thus validating its ability to improve the vertical resolution of the CSERT system.

  16. TIME-LAPSE MODELING AND INVERSION OF CO2 SATURATION FOR SEQUESTRATION AND ENHANCED OIL RECOVERY

    SciTech Connect

    Mark A. Meadows

    2005-05-25

    In this quarter we have continued our development of the inversion procedure for Phase III, in which time-lapse changes in seismic attributes are inverted to yield changes in CO{sub 2} fluid properties over time. In order to extract seismic attributes from the Sleipner North Sea CO{sub 2} time-lapse data set, a new, detailed interpretation was performed and multiple horizons were picked for the 1994 and 2002 vintages. Traveltime difference maps were constructed at several levels within the Sleipner CO{sub 2} injection zone, and were quantitatively analyzed. No traveltime change was seen in the overburden, as expected, while significant changes were seen in the upper half of the CO{sub 2} injection zone. Evidence of thin-bed tuning and undershooting was also found. A new semi-automated, quantitative method for estimating time sag anomalies was developed, and was used to calculate the amount of time sag along a selected horizon. The resulting time sag estimates matched those seen in the traveltime difference maps. Such a method will be useful for obtaining rapid, accurate quantitative measurements of traveltime changes in the Sleipner data cubes. The traveltime changes will be combined with other attributes, such as amplitude and frequency changes, for input into the real-data inversion.

  17. A cautionary note against embryo aneuploidy risk assessment using time-lapse imaging.

    PubMed

    Ottolini, Christian; Rienzi, Laura; Capalbo, Antonio

    2014-03-01

    Preimplantation genetic screening (PGS) for embryo aneuploidy using embryo biopsy is a widely available technique used to select embryos for transfer following IVF for certain patient populations. Since its introduction, there has been an ongoing search for a non-invasive technique to perform PGS. Such an advance would revolutionize the field of IVF enabling PGS to be used universally as a routine embryo selection tool with the potential to significantly increase pregnancy rates and decrease poor outcomes such as miscarriage. Recent publications illustrating the development of an algorithm using time-lapse imaging of IVF embryos have claimed to have done just this. We believe that the statements made in these articles, which include the proposed ability to increase pregnancy rates by determining embryo aneuploidy risk by time-lapse imaging, are premature and to this point unsubstantiated by the published data. We provide evidence from existing publications and from our own data that suggests that the statements recently made are misleading. We make the point that further investigation is needed either in the form of a larger, age-adjusted data set or preferably in a randomized controlled trial.

  18. The time-lapse AVO difference inversion for changes in reservoir parameters

    NASA Astrophysics Data System (ADS)

    Longxiao, Zhi; Hanming, Gu; Yan, Li

    2016-12-01

    The result of conventional time-lapse seismic processing is the difference between the amplitude and the post-stack seismic data. Although stack processing can improve the signal-to-noise ratio (SNR) of seismic data, it also causes a considerable loss of important information about the amplitude changes and only gives the qualitative interpretation. To predict the changes in reservoir fluid more precisely and accurately, we also need the quantitative information of the reservoir. To achieve this aim, we develop the method of time-lapse AVO (amplitude versus offset) difference inversion. For the inversion of reservoir changes in elastic parameters, we apply the Gardner equation as the constraint and convert the three-parameter inversion of elastic parameter changes into a two-parameter inversion to make the inversion more stable. For the inversion of variations in the reservoir parameters, we infer the relation between the difference of the reflection coefficient and variations in the reservoir parameters, and then invert reservoir parameter changes directly. The results of the theoretical modeling computation and practical application show that our method can estimate the relative variations in reservoir density, P-wave and S-wave velocity, calculate reservoir changes in water saturation and effective pressure accurately, and then provide reference for the rational exploitation of the reservoir.

  19. Tracking CO2 Plume in Deep Saline Formations Utilizing a Time-lapse Pressure Tomography Approach

    NASA Astrophysics Data System (ADS)

    Hu, L.; Bayer, P.; Brauchler, R.

    2015-12-01

    CO2 storage in deep saline formations is considered as an attractive option to cut down greenhouse gas emissions. Among the major challenges is the development of efficient technologies for controlling and monitoring the evolution of CO2 plumes during and after injection in the underground. As an alternative to the most commonly used geophysical approaches for subsurface characterization, we propose a pressure-based tomographical approach to track CO2 plume history. By taking into account the direct relationship between saturation and flow properties, pressure tomography has the potential not only to detect a plume but also to estimate the saturation of CO2. The experimental set-up of pressure tomography involves injection of brine or CO2 at variable depths (sources). We use a time-lapse approach, considering first the CO2-free formation, and then the multi-phase CO2-brine system. By applying a rapid eikonal-based inversion technique, pressure fluctuations at observation locations (receivers) are utilized to reconstruct the spatial distribution of the apparent single-phase and mixed-phase diffusivity. Evolution of the plume shape is then delineated by comparison of diffusivity tomograms derived from different times. Finally, an integrated value of CO2 saturation within the plume is obtained by means of a single-phase proxy. Applicability of this novel approach is evaluated in different virtual formations. The time-lapse pressure tomographic investigation revealed that knowledge about the spatial heterogeneity of permeability has a remarkable impact on proper characterization of plume shape.

  20. The Extreme Ice Survey: Capturing and Conveying Glacial Processes Through Time-Lapse Imagery and Narration

    NASA Astrophysics Data System (ADS)

    Balog, J. D.; Box, J. E.; Pfeffer, W. T.; Hood, E. W.; Fagre, D. B.; Anker, C.; O'Neel, S.

    2010-12-01

    The Extreme Ice Survey (EIS) uses time-lapse photography, conventional photography, and video to document rapid change in the Earth's glacial ice. The EIS team currently has 38 time-lapse cameras at sites in Greenland, Iceland, Alaska, the Rocky Mountains and Nepal. EIS supplements this ongoing record with annual repeat photography in British Columbia, Iceland, the Alps, and Bolivia. EIS imagery supplies basic knowledge in glacier dynamics to the science community, as well as compelling, engaging narratives to the general public about the immediacy of the Anthropocene and climate change. Visual materials from EIS have impacted more than 150 million people, ranging from White House staff, the U. S. Congress and government agency officials to globally influential corporate officers and all age strata of the general public. Media products include a National Geographic/NOVA special, two National Geographic magazine articles, a feature in Parade magazine (circulation 71 million), and numerous presentations on CNN, NBC, BBC and National Public Radio. Columbia Glacier, Alaska, June 2006, May 2007, June 2008 terminus indicated.

  1. Database for temporal events and spatial object features in time-lapse images

    NASA Astrophysics Data System (ADS)

    Eggers, Charles E.; Trivedi, Mohan M.

    2000-04-01

    We present an image database system with the capability to locate specified object-level merge and separation events in a sequence of time-lapse images. Specifically, the objects of interest are live cells in phase contrast images acquired by scanning cytometry. The system is named TERSIS and it resides on a workstation accessing time lapse images on CD- ROM. The cell objects are segmented and the resulting data are processed to extract a time series and its time derivative series for each spatial feature. Cell objects are tracked through the image sequence by applying similarity metrics to the cell object feature vectors, and cell merge and separation events are located using global image statistics. Multiple hypotheses are generated and scored to determine participating cell objects in merge/separation events. The cell association and time-varying spatial data re stored in a database. A graphical suer interface provides the user with tools to specify queries for specific cellular states and events for recall and display. Primary limitation include the need for an automatic front-end segmenter and increased cell tracking volume. The design of this system is extensible to other object types and forms of sequential image input, including video.

  2. Very-high-resolution time-lapse photography for plant and ecosystems research1

    PubMed Central

    Nichols, Mary H.; Steven, Janet C.; Sargent, Randy; Dille, Paul; Schapiro, Joshua

    2013-01-01

    • Premise of the study: Traditional photography is a compromise between image detail and area covered. We report a new method for creating time-lapse sequences of very-high-resolution photographs to produce zoomable images that facilitate observation across a range of spatial and temporal scales. • Methods and Results: A robotic camera mount and software were used to capture images of the growth and movement in Brassica rapa every 15 s in the laboratory. The resultant time-lapse sequence (http://timemachine.gigapan.org/wiki/Plant_Growth) captures growth detail such as circumnutation. A modified, solar-powered system was deployed at a remote field site in southern Arizona. Images were collected every 2 h over a 3-mo period to capture the response of vegetation to monsoon season rainfall (http://timemachine.gigapan.org/wiki/Arizona_Grasslands). • Conclusions: A technique for observing time sequences of both individual plant and ecosystem response at a range of spatial scales is available for use in the laboratory and in the field. PMID:25202588

  3. Time-Lapse Electrical Resistivity Investigations for Imaging the Grouting Injection in Shallow Subsurface Cavities

    PubMed Central

    Farooq, Muhammad; Kim, Jung Ho; Song, Young Soo; Amjad Sabir, Mohammad; Umar, Muhammad; Tariq, Mohammad; Muhammad, Said

    2014-01-01

    The highway of Yongweol-ri, Muan-gun, south-western part of the South Korean Peninsula, is underlain by the abandoned of subsurface cavities, which were discovered in 2005. These cavities lie at shallow depths with the range of 5∼15 meters below the ground surface. Numerous subsidence events have repeatedly occurred in the past few years, damaging infrastructure and highway. As a result of continuing subsidence issues, the Korean Institute of Geosciences and Mineral Resources (KIGAM) was requested by local administration to resolve the issue. The KIGAM used geophysical methods to delineate subsurface cavities and improve more refined understanding of the cavities network in the study area. Cement based grouting has been widely employed in the construction industry to reinforce subsurface ground. In this research work, time-lapse electrical resistivity surveys were accomplished to monitor the grouting injection in the subsurface cavities beneath the highway, which have provided a quasi-real-time monitoring for modifying the subsurface cavities related to ground reinforcement, which would be difficult with direct methods. The results obtained from time-lapse electrical resistivity technique have satisfactory imaged the grouting injection experiment in the subsurface cavities beneath the highway. Furthermore, the borehole camera confirmed the presence of grouting material in the subsurface cavities, and hence this procedure increases the mechanical resistance of subsurface cavities below the highway. PMID:24578621

  4. High-resolution, time-lapse imaging for ecosystem-scale phenotyping in the field.

    PubMed

    Brown, Tim; Zimmermann, Christopher; Panneton, Whitney; Noah, Nina; Borevitz, Justin

    2012-01-01

    The high spatial and temporal resolution of data required for high-throughput phenotyping has typically been all but impossible to obtain in field populations of plants. When studies of individual and population genetic variation and microclimate sensor data are combined with phenology data, a landscape-level view of how populations respond to changing environments can be obtained. This chapter will discuss the development of a multi-billion pixel ("gigapixel") camera system that enables the collection of phenology data at up to hourly intervals from in situ plant populations. Such gigapixel time-lapse imaging systems represent a key technological advancement for enabling high-throughput phenotyping in field settings. Gigapixel resolution image datasets allow researchers to record life-history (phenology) data across an entire landscape over multiple seasons. Image data can be wirelessly transmitted to a remote server where it can be accessed online within hours of capture. The time-lapse panoramic images are browsable through an interactive web tool that can be used to compare plant phenology with environmental sensor data collected simultaneously from the field. The high spatial and temporal resolution data can be used to identify individual plant phenology, which can in turn be used to generate complete population level phenotype data. The Gigavision platform is especially powerful when coupled with next-generation population genomic analysis. The Gigavision system permits the rapid identification of the phenotypes and genotypes responding to natural selection in wild populations.

  5. Time-lapse electrical resistivity investigations for imaging the grouting injection in shallow subsurface cavities.

    PubMed

    Farooq, Muhammad; Park, Samgyu; Kim, Jung Ho; Song, Young Soo; Amjad Sabir, Mohammad; Umar, Muhammad; Tariq, Mohammad; Muhammad, Said

    2014-01-01

    The highway of Yongweol-ri, Muan-gun, south-western part of the South Korean Peninsula, is underlain by the abandoned of subsurface cavities, which were discovered in 2005. These cavities lie at shallow depths with the range of 5∼15 meters below the ground surface. Numerous subsidence events have repeatedly occurred in the past few years, damaging infrastructure and highway. As a result of continuing subsidence issues, the Korean Institute of Geosciences and Mineral Resources (KIGAM) was requested by local administration to resolve the issue. The KIGAM used geophysical methods to delineate subsurface cavities and improve more refined understanding of the cavities network in the study area. Cement based grouting has been widely employed in the construction industry to reinforce subsurface ground. In this research work, time-lapse electrical resistivity surveys were accomplished to monitor the grouting injection in the subsurface cavities beneath the highway, which have provided a quasi-real-time monitoring for modifying the subsurface cavities related to ground reinforcement, which would be difficult with direct methods. The results obtained from time-lapse electrical resistivity technique have satisfactory imaged the grouting injection experiment in the subsurface cavities beneath the highway. Furthermore, the borehole camera confirmed the presence of grouting material in the subsurface cavities, and hence this procedure increases the mechanical resistance of subsurface cavities below the highway.

  6. Time-lapse nanoscopy of friction in the non-Amontons and non-Coulomb regime.

    PubMed

    Ishida, Tadashi; Sato, Takaaki; Ishikawa, Takahiro; Oguma, Masatsugu; Itamura, Noriaki; Goda, Keisuke; Sasaki, Naruo; Fujita, Hiroyuki

    2015-03-11

    Originally discovered by Leonard da Vinci in the 15th century, the force of friction is directly proportional to the applied load (known as Amontons' first law of friction). Furthermore, kinetic friction is independent of the sliding speed (known as Coulomb's law of friction). These empirical laws break down at high normal pressure (due to plastic deformation) and low sliding speed (in the transition regime between static friction and kinetic friction). An important example of this phenomenon is friction between the asperities of tectonic plates on the Earth. Despite its significance, little is known about the detailed mechanism of friction in this regime due to the lack of experimental methods. Here we demonstrate in situ time-lapse nanoscopy of friction between asperities sliding at ultralow speed (∼0.01 nm/s) under high normal pressure (∼GPa). This is made possible by compressing and rubbing a pair of nanometer-scale crystalline silicon anvils with electrostatic microactuators and monitoring its dynamical evolution with a transmission electron microscope. Our analysis of the time-lapse movie indicates that superplastic behavior is induced by decrystallization, plastic deformation, and atomic diffusion at the asperity-asperity interface. The results hold great promise for a better understanding of quasi-static friction under high pressure for geoscience, materials science, and nanotechnology.

  7. Coda-wave interferometry analysis of time-lapse VSP data for monitoring geological carbon sequestration

    SciTech Connect

    Zhou, R.; Huang, L.; Rutledge, J.T.; Fehler, M.; Daley, T.M.; Majer, E.L.

    2009-11-01

    Injection and movement/saturation of carbon dioxide (CO2) in a geological formation will cause changes in seismic velocities. We investigate the capability of coda-wave interferometry technique for estimating CO2-induced seismic velocity changes using time-lapse synthetic vertical seismic profiling (VSP) data and the field VSP datasets acquired for monitoring injected CO2 in a brine aquifer in Texas, USA. Synthetic VSP data are calculated using a finite-difference elastic-wave equation scheme and a layered model based on the elastic Marmousi model. A possible leakage scenario is simulated by introducing seismic velocity changes in a layer above the CO2 injection layer. We find that the leakage can be detected by the detection of a difference in seismograms recorded after the injection compared to those recorded before the injection at an earlier time in the seismogram than would be expected if there was no leakage. The absolute values of estimated mean velocity changes, from both synthetic and field VSP data, increase significantly for receiver positions approaching the top of a CO2 reservoir. Our results from field data suggest that the velocity changes caused by CO2 injection could be more than 10% and are consistent with results from a crosswell tomogram study. This study demonstrates that time-lapse VSP with coda-wave interferometry analysis can reliably and effectively monitor geological carbon sequestration.

  8. Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing

    PubMed Central

    Pang, Yonggang; Tsigkou, Olga; Spencer, Joel A.; Lin, Charles P.; Neville, Craig

    2015-01-01

    Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (d,l-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad

  9. Design of novel 3D gene activated PEG scaffolds with ordered pore structure.

    PubMed

    Orsi, Silvia; Guarnieri, Daniela; Netti, Paolo A

    2010-03-01

    The ability to genetically modify cells seeded inside synthetic hydrogel scaffolds offers a suitable approach to induce and control tissue repair and regeneration guiding cell fate. In fact the transfected cells can act as local in vivo bioreactor, secreting plasmid encoded proteins that augment tissue regeneration processes. We have realized a DNA bioactivated high porous poly(ethylene glycol) (PEG) matrix by polyethyleneimine (PEI)/DNA complexes adsorption. As the design of the microarchitectural features of a scaffold also contributes to promote and influence cell fate, we appropriately designed the inner structure of gene activated PEG hydrogels by gelatine microparticles templating. Microarchitectural properties of the scaffold were analysed by scanning electron microscopy. 3D cell migration and transfection were monitored through time-lapse videomicroscopy and confocal laser scanning microscopy.

  10. Near-wall 3D velocity measurements above biomimetic shark skin denticles using Digital In-line Holographic Microscopy

    NASA Astrophysics Data System (ADS)

    Toloui, Mostafa; Brajkovic, David; Hong, Jiarong

    2014-11-01

    Digital In-line Holography is employed to image 3D flow structures in the vicinity of a transparent rough surface consisting of closely packed biomimetic shark skin denticles as roughness elements. The 3D printed surface replicates the morphological features of real shark skin, and the denticles have a geometrical scale of 2 mm, i.e. 10 times of the real ones. In order to minimize optical aberrations near the fluid-roughness interface and enable flow measurements around denticles, the optical refractive index of the fluid medium is maintained the same as that of the denticle model in an index-matched flow facility using NaI solution as the working fluid. The experiment is conducted in a 1.2 m long test section with 50 mm × 50 mm cross section. The sampling volume is located in the downstream region of a shark skin replica of 12'' stretch where the turbulent flow is fully-developed and the transitional effect from smooth to the rough surface becomes negligible. Several instantaneous realizations of the 3D velocity field are obtained and are used to illustrate turbulent coherent structures induced by shark-skin denticles. This information will provide insights on the hydrodynamic function of shark's unique surface ornamentation.

  11. Time-lapse borehole radar for monitoring rainfall infiltration through podosol horizons in a sandy vadose zone

    NASA Astrophysics Data System (ADS)

    Strobach, Elmar; Harris, B. D.; Dupuis, J. C.; Kepic, A. W.

    2014-03-01

    The shallow aquifer on the Gnangara Mound, north of Perth, Western Australia, is recharged by winter rainfall. Water infiltrates through a sandy Podosol where cemented accumulation (B-) horizons are common. They are water retentive and may impede recharge. To observe wetting fronts and the influence of soil horizons on unsaturated flow, we deployed time-lapse borehole radar techniques sensitive to soil moisture variations during an annual recharge cycle. Zero-offset crosswell profiling (ZOP) and vertical radar profiling (VRP) measurements were performed at six sites on a monthly basis before, during, and after annual rainfall in 2011. Water content profiles are derived from ZOP logs acquired in closely spaced wells. Sites with small separation between wells present potential repeatability and accuracy difficulties. Such problems could be lessened by (i) ZOP saturated zone velocity matching of time-lapse curves, and (ii) matching of ZOP and VRP results. The moisture contents for the baseline condition and subsequent observations are computed using the Topp relationship. Time-lapse moisture curves reveal characteristic vadose zone infiltration regimes. Examples are (I) full recharge potential after 200 mm rainfall, (II) delayed wetting and impeded recharge, and (III) no recharge below 7 m depth. Seasonal infiltration trends derived from long-term time-lapse neutron logging at several sites are shown to be comparable with infiltration trends recovered from time-lapse crosswell radar measurements. However, radar measurements sample a larger volume of earth while being safer to deploy than the neutron method which employs a radioactive source. For the regime (III) site, where time-lapse radar indicates no net recharge or zero flux to the water table, a simple water balance provides an evapotranspiration value of 620 mm for the study period. This value compares favorably to previous studies at similar test sites in the region. Our six field examples demonstrate

  12. Time-lapse VSP data processing for monitoring CO2 injection

    SciTech Connect

    Huang, Lianjie; Rutledge, James; Cheng, Arthur

    2009-01-01

    As a part of the effort of the Southwest Regional Partnership on Carbon Sequestration supported by U.S. Department of Energy and managed by the National Energy Technology Laboratory, two sets of time-lapse VSPs were acquired and processed in oil fields undergoing CO{sub 2} injection. One set of VSPs was acquired at the Aneth oil field in Utah, the other set at the Scurry Area Canyon Reef Operators Committee (SACROC) field in West Texas. One baseline and two repeat VSP surveys were conducted from 2007 to 2009 at the Aneth oil field in Utah for monitoring CO{sub 2} injection. The aim of the time-lapse VSP surveys is to study the combined enhanced oil recovery (EOR) and CO{sub 2} sequestration in collaboration with Resolute Natural Resources, Inc. VSP data were acquired using a cemented geophone string with 60 levels at depth from 805 m to 1704 m, and CO{sub 2} is injected into a horizontal well nearby within the reservoir at depth approximately from 1730 m to 1780 m. For each VSP survey, the data were acquired for one zero-offset source location and seven offset source locations (Figure 1). The baseline VSP survey was conducted before the CO{sub 2} injection. More than ten thousand tons of CO{sub 2} was injected between each of the two repeat VSP surveys. There are three horizontal injection wells, all originating from the same vertical well. One is drilled towards Southeast, directly towards the monitoring well (Figure 2), and the other two towards Northwest, directly away from the monitoring well. The injection is into the top portion of the Desert Creek formation, just beneath the Gothic shale, which acts as the reservoir seal. The initial baseline acquisition was done in October 2007; subsequent time-lapse acquisitions were conducted in July 2008, and January 2009. The acquisition geometry is shown in Figure 1. Shot point 1 is the zero-offset source location, Shot points 2 to 8 are the seven offset VSPs, arranged in a quarter circle on the Northwest side of the

  13. Active and passive electrical and seismic time-lapse monitoring of earthen embankments

    NASA Astrophysics Data System (ADS)

    Rittgers, Justin Bradley

    In this dissertation, I present research involving the application of active and passive geophysical data collection, data assimilation, and inverse modeling for the purpose of earthen embankment infrastructure assessment. Throughout the dissertation, I identify several data characteristics, and several challenges intrinsic to characterization and imaging of earthen embankments and anomalous seepage phenomena, from both a static and time-lapse geophysical monitoring perspective. I begin with the presentation of a field study conducted on a seeping earthen dam, involving static and independent inversions of active tomography data sets, and self-potential modeling of fluid flow within a confined aquifer. Additionally, I present results of active and passive time-lapse geophysical monitoring conducted during two meso-scale laboratory experiments involving the failure and self-healing of embankment filter materials via induced vertical cracking. Identified data signatures and trends, as well as 4D inversion results, are discussed as an underlying motivation for conducting subsequent research. Next, I present a new 4D acoustic emissions source localization algorithm that is applied to passive seismic monitoring data collected during a full-scale embankment failure test. Acoustic emissions localization results are then used to help spatially constrain 4D inversion of collocated self-potential monitoring data. I then turn to time-lapse joint inversion of active tomographic data sets applied to the characterization and monitoring of earthen embankments. Here, I develop a new technique for applying spatiotemporally varying structural joint inversion constraints. The new technique, referred to as Automatic Joint Constraints (AJC), is first demonstrated on a synthetic 2D joint model space, and is then applied to real geophysical monitoring data sets collected during a full-scale earthen embankment piping-failure test. Finally, I discuss some non-technical issues related to

  14. Application of Time-Lapse Seismic Monitoring for the Control and Optimization of CO2 Enhanced Oil Recovery Operations

    SciTech Connect

    Brian Toelle

    2008-11-30

    This project, 'Application of Time-Lapse Seismic Monitoring for the Control and Optimization of CO{sub 2} Enhanced Oil Recovery Operations', investigated the potential for monitoring CO{sub 2} floods in carbonate reservoirs through the use of standard p-wave seismic data. This primarily involved the use of 4D seismic (time lapse seismic) in an attempt to observe and map the movement of the injected CO{sub 2} through a carbonate reservoir. The differences between certain seismic attributes, such as amplitude, were used for this purpose. This technique has recently been shown to be effective in CO{sub 2} monitoring in Enhanced Oil Recovery (EOR) projects, such as Weyborne. This study was conducted in the Charlton 30/31 field in the northern Michigan Basin, which is a Silurian pinnacle reef that completed its primary production in 1997 and was scheduled for enhanced oil recovery using injected CO{sub 2}. Prior to injection an initial 'Base' 3D survey was obtained over the field and was then processed and interpreted. CO{sub 2} injection within the main portion of the reef was conducted intermittently during 13 months starting in August 2005. During this time, 29,000 tons of CO{sub 2} was injected into the Guelph formation, historically known as the Niagaran Brown formation. By September 2006, the reservoir pressure within the reef had risen to approximately 2000 lbs and oil and water production from the one producing well within the field had increased significantly. The determination of the reservoir's porosity distribution, a critical aspect of reservoir characterization and simulation, proved to be a significant portion of this project. In order to relate the differences observed between the seismic attributes seen on the multiple 3D seismic surveys and the actual location of the CO{sub 2}, a predictive reservoir simulation model was developed based on seismic attributes obtained from the base 3D seismic survey and available well data. This simulation predicted

  15. Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

    PubMed

    Sivaguru, Mayandi; Fried, Glenn; Sivaguru, Barghav S; Sivaguru, Vignesh A; Lu, Xiaochen; Choi, Kyung Hwa; Saif, M Taher A; Lin, Brian; Sadayappan, Sakthivel

    2015-11-01

    The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

  16. Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging.

    PubMed

    Miwa, Shinji; Yano, Shuya; Kimura, Hiroaki; Yamamoto, Mako; Toneri, Makoto; Murakami, Takashi; Hayashi, Katsuhiro; Yamamoto, Norio; Fujiwara, Toshiyoshi; Tsuchiya, Hiroyuki; Hoffman, Robert M

    2015-01-01

    The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m(2) UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G1/S transition were analyzed using the Kaplan-Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G2/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m(2) UVB during G0/G1 phase had a higher survival rate than the cells irradiated during S/G2/M phase. A minority of cells could escape S/G2/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G1/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m(2) resulted in a greater number of apoptotic cells.

  17. Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging

    PubMed Central

    Saalfrank, Dirk; Konduri, Anil Krishna; Latifi, Shahrzad; Habibey, Rouhollah; Golabchi, Asiyeh; Martiniuc, Aurel Vasile; Knoll, Alois; Ingebrandt, Sven; Blau, Axel

    2015-01-01

    Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes. PMID:26543581

  18. 3-D confocal laser scanning microscopy used in morphometric analysis of rat Purkinje cell dendritic spines after chronic ethanol consumption.

    PubMed

    Wenisch, S; Fortmann, B; Steinmetz, T; Kriete, A; Leiser, R; Bitsch, I

    1998-12-01

    A confocal laser scanning microscope (with a 543 nm laser) was used for imaging rat Purkinje cell dendritic spines at high 3-D resolution. In a nutritionally controlled study of the rat, 5 months of ethanol consumption was demonstrated to alter the spines of Purkinje cell dendrites in rat cerebellum. Intact spines showed significant elongation after ethanol exposure, whereas this neuromorphological alteration could not be detected in controls. Spine elongation could be regarded as compensative growth of spines in search of new synaptic contacts due to alcohol induced cell loss.

  19. A 3D imaging and visualization workflow, using confocal microscopy and advanced image processing for brachyuran crab larvae.

    PubMed

    Kamanli, S A; Kihara, T C; Ball, A D; Morritt, D; Clark, P F

    2017-03-07

    Confocal laser scanning microscopy is an excellent tool for nondestructive imaging of arthropods and can provide detailed information on morphology including fine surface detail. A methodology is presented here for the visualization by confocal microscopy of arthropods, using brachyuran crab zoeal stages as examples and postprocessing techniques derived from micro-CT protocols to improve the final images. This protocol is divided into description of the preprocessing steps (cleaning, staining, digesting and mounting), confocal laser scanning microscopy and data visualization using open-source, freeware programs ImageJ and Drishti. The advantages of using ImageJ to standardize stack data and Drishti for surface rendering are discussed. The methodology has been comprehensively tested using data acquired from all four brands of confocal microscope (Leica, Nikon, Olympus and Zeiss).

  20. Time-lapse cinematography of the capillary tube cell migration inhibition test.

    PubMed

    Bray, M A

    1980-01-01

    The kinetics of human and guinea pig cell migration inhibition have been studied using time-lapse cinematography of cells migrating from capillary tubes. Guinea pig and human cells exhibit markedly different kinetics in the absence of inhibitors. Specific antigen causes a dose-related inhibition of migration for up to 60 h using guinea pig cells and a peak of inhibition after 18 h using the human leucocyte system. The timing of measurement of maximum activity more critical for the latter test. The kinetics of lymphokine generation have been examined and the migration inhibitory activity of the plant mitogen (PHA), a Kurloff cell product and a continuous cell line supernatant have been compared with the inhibitory profiles of lymphokine preparations and specific antigen.

  1. Electron microscopic time-lapse visualization of surface pore filtration on particulate matter trapping process.

    PubMed

    Sanui, Ryoko; Hanamura, Katsunori

    2016-09-01

    A scanning electron microscope (SEM) was used to dynamically visualize the particulate matter (PM) trapping process on diesel particulate filter (DPF) walls at a micro scale as 'time-lapse' images corresponding to the increase in pressure drop simultaneously measured through the DPF. This visualization and pressure drop measurement led to the conclusion that the PM trapping in surface pores was driven by PM bridging and stacking at constricted areas in porous channels. This caused a drastic increase in the pressure drop during PM accumulation at the beginning of the PM trapping process. The relationship between the porous structure of the DPF and the depth of the surface pore was investigated in terms of the porosity distribution and PM penetration depth near the wall surface with respect to depth. The pressure drop calculated with an assumed surface pore depth showed a good correspondence to the measured pressure drop.

  2. A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution

    SciTech Connect

    Hirano, Masahiko; Hoshida, Tetsushi; Sakaue-Sawano, Asako; Miyawaki, Atsushi

    2010-02-12

    We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO{sub 2}-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.

  3. Visualizing and quantifying movement from pre-recorded videos: The spectral time-lapse (STL) algorithm.

    PubMed

    Madan, Christopher R; Spetch, Marcia L

    2014-01-01

    When studying animal behaviour within an open environment, movement-related data are often important for behavioural analyses. Therefore, simple and efficient techniques are needed to present and analyze the data of such movements. However, it is challenging to present both spatial and temporal information of movements within a two-dimensional image representation. To address this challenge, we developed the spectral time-lapse (STL) algorithm that re-codes an animal's position at every time point with a time-specific color, and overlays it with a reference frame of the video, to produce a summary image. We additionally incorporated automated motion tracking, such that the animal's position can be extracted and summary statistics such as path length and duration can be calculated, as well as instantaneous velocity and acceleration. Here we describe the STL algorithm and offer a freely available MATLAB toolbox that implements the algorithm and allows for a large degree of end-user control and flexibility.

  4. Time-Lapse Electrical Resistivity Monitoring of the Remediation of a Saline Groundwater Plume

    NASA Astrophysics Data System (ADS)

    Hayley, K.; Bentlay, L. R.; Gharibi, M.

    2007-12-01

    Time-lapse electrical resistivity (ER) surveys are used to monitor the movement and remediation of a saline contaminant plume over the span of three years. Temperature is responsible for a large proportion of the observed ER changes. A temperature resistivity relationship is developed through laboratory testing on core from the site and analysis of petrophysical models. We present a protocol for removing the effects of temperature from ER difference images. Saturation changes also contribute to changes in the ER images. However, at this site, saturation is too spatially heterogeneous to apply a simple correction as was done with temperature. We show that the magnitude of the saturation changes are low and present an analysis of the uncertainty this introduces into the image interpretation. After accounting for temperature variations, we observe changes in the subsurface ER distribution that are consistent with depression focused recharge, solute flushing, and spatially variable effectiveness of the remediation program.

  5. TRIIG - Time-lapse reproduction of images through interactive graphics. [digital processing of quality hard copy

    NASA Technical Reports Server (NTRS)

    Buckner, J. D.; Council, H. W.; Edwards, T. R.

    1974-01-01

    Description of the hardware and software implementing the system of time-lapse reproduction of images through interactive graphics (TRIIG). The system produces a quality hard copy of processed images in a fast and inexpensive manner. This capability allows for optimal development of processing software through the rapid viewing of many image frames in an interactive mode. Three critical optical devices are used to reproduce an image: an Optronics photo reader/writer, the Adage Graphics Terminal, and Polaroid Type 57 high speed film. Typical sources of digitized images are observation satellites, such as ERTS or Mariner, computer coupled electron microscopes for high-magnification studies, or computer coupled X-ray devices for medical research.

  6. Cell invasion with proliferation mechanisms motivated by time-lapse data

    NASA Astrophysics Data System (ADS)

    Simpson, Matthew J.; Landman, Kerry A.; Hughes, Barry D.

    2010-09-01

    Cell invasion involves a population of cells which are motile and proliferative. Traditional discrete models of proliferation involve agents depositing daughter agents on nearest-neighbor lattice sites. Motivated by time-lapse images of cell invasion, we propose and analyze two new discrete proliferation models in the context of an exclusion process with an undirected motility mechanism. These discrete models are related to a family of reaction-diffusion equations and can be used to make predictions over a range of scales appropriate for interpreting experimental data. The new proliferation mechanisms are biologically relevant and mathematically convenient as the continuum-discrete relationship is more robust for the new proliferation mechanisms relative to traditional approaches.

  7. Limiting aspects of using geophysical time-lapse measurements for contaminant site monitoring

    NASA Astrophysics Data System (ADS)

    French, H. K.; Bloem, E.

    2010-12-01

    Winter maintenance at airports and roads in the areas with winter frost requires the use of large quantities of de-icing chemicals. These chemicals infiltrate the unsaturated zone during winter and spring due to the mixing with snow next to the roads or runways and may hence pollute the groundwater. Geophysical methods provide insight into soil heterogeneity and characteristics and may, when used in time-lapse mode, serve as a monitoring technique for contaminant transport over larger areas than traditional sampling techniques such as suction cups, soil sampling techniques and groundwater wells. The presence of a mixture of materials and contaminants in the subsurface, as well as the natural temporal variable conditions such as temperature and water saturation are among the challenges of geophysical monitoring of flow and transport processes in the unsaturated zone. Some examples of the use of geophysical measurements for contaminant site monitoring from the literature will be given as well as insight to more specific challenges both practical and scientifically for a case study in Norway. The case study shows results of electrical resistivity measurements along two profiles next to one of the runways at Oslo airport, Gardermoen. One profile is located parallel to the runway and within the zone affected by contaminated snow, while the other set of surface electrodes are installed at an angle from the runway and covers areas both affected and unaffected by de-icing chemicals. In addition to time-lapse electrical resistivity measurements, the soil temperature, volumetric water content and the electrical conductivity of the soil water is measured at 4 depths at the crossing point of the two cables. Theoretical improvements and managerial aspects still required for the applicability of this monitoring technique at contaminated sites will be discussed.

  8. Assessment of Saturation Patterns on Agricultural Land Using Time-lapse Photography

    NASA Astrophysics Data System (ADS)

    Silasari, R.; Bloeschl, G.

    2015-12-01

    Agricultural land generates overland flow differently from natural environment due to features from anthropogenic activities such as cultivated soil layer and tile drain pipe. During rainfall events, the formation of overland flow may happen from infiltration excess and/or saturation excess according to the threshold processes which are influenced by rainfall characteristics and soil hydraulic parameters. The dynamics of threshold processes in varying rainfall and soil hydraulic conditions will affect the surface runoff response which can be inversely analyzed by visually observing the generated saturation patterns. This study aims to explore the use of time-lapse photographs of saturated plot during rainfall events to observe and understand the threshold processes of overland flow generation. The observation was conducted at Hydrological Open Air Laboratory (HOAL) in Lower Austria with a 2 megapixels surveillance camera overlooking a 1.8 ha tile-drained agricultural field situated on a hillslope. The main tile drain pipe extends from the higher ground into the riparian area - creating a depression line which generates the main saturation track. The time-lapse photographs are able to capture the spatial and temporal dynamics of 0.1 ha saturated plot (117 m long and 10 m wide in average) during three big rainfall events in 2014 which produced measurable overland flow. The photographs also manage to capture the behavior of overland flow on tractor tracks which were generated faster than on the main saturated plot - due to the more compacted soil - and contribute significantly to the overall overland flow discharge and movements. Comparison of the photographs with on-field manual plotting shows good accuracy of the captured saturation plot and the possibility of calculating the plot area digitally. This method gives opportunity to observe overland flow generation on visual basis as a complement of the customary discharge measurements.

  9. Improving time-lapse seismic repeatability: CO2CRC Otway site permanent geophone array field trials

    NASA Astrophysics Data System (ADS)

    Pevzner, Roman; Dupuis, Christian; Shulakova, Valeriya; Urosevic, Milovan; Lumley, David

    2013-04-01

    The proposed Stage 2C of the CO2CRC Otway project involves injection of a small amount (around 15,000 tonnes) of CO2/CH4 gas mixture into saline acquifer (Paaratte formation) at the depth of ~1.5 km. The seismic time-lapse signal will depend largely on the formation properties and the injection scenario, but is likely to be relatively weak. In order to improve time-lapse seismic monitoring capabilities by decreasing the noise level, a buried receiver arrays can be used. A small-scale trial of such an array was conducted at Otway site in June 2012. A set of 25 geophones was installed in 3 m deep boreholes in parallel to the same number of surface geophones. In addition, four geophones were placed into boreholes of 1 to 12 m depth. In order to assess the gain in the signal-to-noise ratio and repeatability, both active and passive seismic surveys were carried out. The surveys were conducted in relatively poor weather conditions, with rain, strong wind and thunderstorms increasing the noise level. We found that noise level for buried geophones is on average 20 dB lower compared to the surface ones. Furthermore, the combination of active and passive experiments has allowed us to perform a detailed classification of various noise sources. Acknowledgement The authors acknowledge the funding provided by the Australian government through its CRC program to support this CO2CRC research project. We also acknowledge the CO2CRC's corporate sponsors and the financial assistance provided through Australian National Low Emissions Coal Research and Development (ANLEC R&D). ANLEC R&D is supported by Australian Coal Association Low Emissions Technology Limited and the Australian Government through the Clean Energy Initiative.

  10. Time-Lapse Imaging of Red Blood Cell Invasion by the Rodent Malaria Parasite Plasmodium yoelii

    PubMed Central

    Yahata, Kazuhide; Treeck, Moritz; Culleton, Richard; Gilberger, Tim-Wolf; Kaneko, Osamu

    2012-01-01

    In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum. PMID:23227208

  11. Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy

    NASA Astrophysics Data System (ADS)

    Czamara, K.; Majzner, K.; Selmi, A.; Baranska, M.; Ozaki, Y.; Kaczor, A.

    2017-01-01

    Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators.

  12. Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy

    PubMed Central

    Czamara, K.; Majzner, K.; Selmi, A.; Baranska, M.; Ozaki, Y.; Kaczor, A.

    2017-01-01

    Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators. PMID:28098251

  13. Analysis of nuclear organization with TANGO, software for high-throughput quantitative analysis of 3D fluorescence microscopy images.

    PubMed

    Ollion, Jean; Cochennec, Julien; Loll, François; Escudé, Christophe; Boudier, Thomas

    2015-01-01

    The cell nucleus is a highly organized cellular organelle that contains the genome. An important step to understand the relationships between genome positioning and genome functions is to extract quantitative data from three-dimensional (3D) fluorescence imaging. However, such approaches are limited by the requirement for processing and analyzing large sets of images. Here we present a practical approach using TANGO (Tools for Analysis of Nuclear Genome Organization), an image analysis tool dedicated to the study of nuclear architecture. TANGO is a generic tool able to process large sets of images, allowing quantitative study of nuclear organization. In this chapter a practical description of the software is drawn in order to give an overview of its different concepts and functionalities. This description is illustrated with a precise example that can be performed step-by-step on experimental data provided on the website http://biophysique.mnhn.fr/tango/HomePage.

  14. 3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria

    NASA Astrophysics Data System (ADS)

    Renz, Anja; Renz, Marc; Klütsch, Diana; Deckers-Hebestreit, Gabriele; Börsch, Michael

    2015-03-01

    FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells.

  15. Segmentation and tracking of adherens junctions in 3D for the analysis of epithelial tissue morphogenesis.

    PubMed

    Cilla, Rodrigo; Mechery, Vinodh; Hernandez de Madrid, Beatriz; Del Signore, Steven; Dotu, Ivan; Hatini, Victor

    2015-04-01

    Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT).

  16. Segmentation and Tracking of Adherens Junctions in 3D for the Analysis of Epithelial Tissue Morphogenesis

    PubMed Central

    Cilla, Rodrigo; Mechery, Vinodh; Hernandez de Madrid, Beatriz; Del Signore, Steven; Dotu, Ivan; Hatini, Victor

    2015-01-01

    Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT) PMID:25884654

  17. Application of Time-Lapse Seismic Monitoring for the Control and Optimization of CO2 Enhanced Oil Recovery Operations

    SciTech Connect

    Brian E. Toelle

    2006-02-28

    The ''Application of Time-Lapse Seismic Monitoring for the Control and Optimization of CO{sub 2} Enhanced Oil Recovery Operations'' project is investigating the potential for monitoring CO{sub 2} floods in carbonate reservoirs through the use of standard p-wave seismic data. This project will involve the use of 4D seismic (time lapse seismic) to try to observe the movement of the injected CO{sub 2} through the reservoir. The differences between certain seismic attributes, such as amplitude, will be used to detect and map the movement of CO{sub 2} within the reservoir. This technique has recently been shown to be effective in CO{sub 2} monitoring in EOR projects such as Weyborne. The project is being conducted in the Charlton 30/31 field in northern Michigan Basin which is a Silurian pinnacle reef that has completed its primary production. This field is now undergoing enhanced oil recovery using CO{sub 2}. The CO{sub 2} flood was initiated the end of 2005 when the injection of small amounts of CO{sub 2} begin in the A1 Carbonate. This injection was conducted for 2 months before being temporarily halted in order for pressure measurements to be conducted. The determination of the reservoir's porosity distribution is proving to be a significant portion of this project. In order to relate the differences observed between the seismic attributes seen on the multiple surveys and the actual location of the CO{sub 2}, a predictive reservoir simulation model had to be developed. From this model, an accurate determination of porosity within the carbonate reservoir must be obtained. For this certain seismic attributes have been investigated. The study reservoirs in the Charlton 30/31 field range from 50 to 400 acres in size. The relatively small area to image makes 3-D seismic data acquisition reasonably cost effective. Permeability and porosity vary considerably throughout the reef, thus it is essential to perform significant reservoir characterization and modeling prior to

  18. Time-lapse seismic waveform modelling and attribute analysis using hydromechanical models for a deep reservoir undergoing depletion

    NASA Astrophysics Data System (ADS)

    He, Y.-X.; Angus, D. A.; Blanchard, T. D.; Wang, G.-L.; Yuan, S.-Y.; Garcia, A.

    2016-04-01

    Extraction of fluids from subsurface reservoirs induces changes in pore pressure, leading not only to geomechanical changes, but also perturbations in seismic velocities and hence observable seismic attributes. Time-lapse seismic analysis can be used to estimate changes in subsurface hydromechanical properties and thus act as a monitoring tool for geological reservoirs. The ability to observe and quantify changes in fluid, stress and strain using seismic techniques has important implications for monitoring risk not only for petroleum applications but also for geological storage of CO2 and nuclear waste scenarios. In this paper, we integrate hydromechanical simulation results with rock physics models and full-waveform seismic modelling to assess time-lapse seismic attribute resolution for dynamic reservoir characterization and hydromechanical model calibration. The time-lapse seismic simulations use a dynamic elastic reservoir model based on a North Sea deep reservoir undergoing large pressure changes. The time-lapse seismic traveltime shifts and time strains calculated from the modelled and processed synthetic data sets (i.e. pre-stack and post-stack data) are in a reasonable agreement with the true earth models, indicating the feasibility of using 1-D strain rock physics transform and time-lapse seismic processing methodology. Estimated vertical traveltime shifts for the overburden and the majority of the reservoir are within ±1 ms of the true earth model values, indicating that the time-lapse technique is sufficiently accurate for predicting overburden velocity changes and hence geomechanical effects. Characterization of deeper structure below the overburden becomes less accurate, where more advanced time-lapse seismic processing and migration is needed to handle the complex geometry and strong lateral induced velocity changes. Nevertheless, both migrated full-offset pre-stack and near-offset post-stack data image the general features of both the overburden and

  19. Selected time-lapse movies of the east rift zone eruption of KĪlauea Volcano, 2004–2008

    USGS Publications Warehouse

    Orr, Tim R.

    2011-01-01

    Since 2004, the U.S. Geological Survey's Hawaiian Volcano Observatory has used mass-market digital time-lapse cameras and network-enabled Webcams for visual monitoring and research. The 26 time-lapse movies in this report were selected from the vast collection of images acquired by these camera systems during 2004–2008. Chosen for their content and broad aesthetic appeal, these image sequences document a variety of flow-field and vent processes from Kīlauea's east rift zone eruption, which began in 1983 and is still (as of 2011) ongoing.

  20. Automated 3D detection and classification of Giardia lamblia cysts using digital holographic microscopy with partially coherent source

    NASA Astrophysics Data System (ADS)

    El Mallahi, A.; Detavernier, A.; Yourassowsky, C.; Dubois, F.

    2012-06-01

    Over the past century, monitoring of Giardia lamblia became a matter of concern for all drinking water suppliers worldwide. Indeed, this parasitic flagellated protozoan is responsible for giardiasis, a widespread diarrhoeal disease (200 million symptomatic individuals) that can lead immunocompromised individuals to death. The major difficulty raised by Giardia lamblia's cyst, its vegetative transmission form, is its ability to survive for long periods in harsh environments, including the chlorine concentrations and treatment duration used traditionally in water disinfection. Currently, there is a need for a reliable, inexpensive, and easy-to-use sensor for the identification and quantification of cysts in the incoming water. For this purpose, we investigated the use of a digital holographic microscope working with partially coherent spatial illumination that reduces the coherent noise. Digital holography allows one to numerically investigate a volume by refocusing the different plane of depth of a hologram. In this paper, we perform an automated 3D analysis that computes the complex amplitude of each hologram, detects all the particles present in the whole volume given by one hologram and refocuses them if there are out of focus using a refocusing criterion based on the integrated complex amplitude modulus and we obtain the (x,y,z) coordinates of each particle. Then the segmentation of the particles is processed and a set of morphological and textures features characteristic to Giardia lamblia cysts is computed in order to classify each particles in the right classes.

  1. In vivo tissue has non-linear rheological behavior distinct from 3D biomimetic hydrogels, as determined by AMOTIV microscopy.

    PubMed

    Blehm, Benjamin H; Devine, Alexus; Staunton, Jack R; Tanner, Kandice

    2016-03-01

    Variation in matrix elasticity has been shown to determine cell fate in both differentiation and development of malignant phenotype. The tissue microenvironment provides complex biochemical and biophysical signals in part due to the architectural heterogeneities found in extracellular matrices (ECMs). Three dimensional cell cultures can partially mimic in vivo tissue architecture, but to truly understand the role of viscoelasticity on cell fate, we must first determine in vivo tissue mechanical properties to improve in vitro models. We employed Active Microrheology by Optical Trapping InVivo (AMOTIV), using in situ calibration to measure in vivo zebrafish tissue mechanics. Previously used trap calibration methods overestimate complex moduli by ∼ 2-20 fold compared to AMOTIV. Applying differential microscale stresses and strains showed that hyaluronic acid (HA) gels display semi-flexible polymer behavior, while laminin-rich ECM hydrogels display flexible polymer behavior. In contrast, zebrafish tissues displayed different moduli at different stresses, with higher power law exponents at lower stresses, indicating that living tissue has greater stress dependence than the 3D hydrogels examined. To our knowledge, this work is the first vertebrate tissue rheological characterization performed in vivo. Our fundamental observations are important for the development and refinement of in vitro platforms.

  2. High-Resolution 3D Imaging and Quantification of Gold Nanoparticles in a Whole Cell Using Scanning Transmission Ion Microscopy

    PubMed Central

    Chen, Xiao; Chen, Ce-Belle; Udalagama, Chammika N.B.; Ren, Minqin; Fong, Kah Ee; Yung, Lin Yue Lanry; Giorgia, Pastorin; Bettiol, Andrew Anthony; Watt, Frank

    2013-01-01

    Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution. PMID:23561518

  3. Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

    NASA Astrophysics Data System (ADS)

    Pena, Ana-Maria; Fagot, Dominique; Olive, Christian; Michelet, Jean-François; Galey, Jean-Baptiste; Leroy, Frédéric; Beaurepaire, Emmanuel; Martin, Jean-Louis; Colonna, Anne; Schanne-Klein, Marie-Claire

    2010-09-01

    Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

  4. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  5. Calibration-free absolute quantification of optical absorption coefficients using acoustic spectra in 3D photoacoustic microscopy of biological tissue.

    PubMed

    Guo, Zijian; Hu, Song; Wang, Lihong V

    2010-06-15

    Optical absorption is closely associated with many physiological important parameters, such as the concentration and oxygen saturation of hemoglobin, and it can be used to quantify the concentrations of nonfluorescent molecules. We propose a method to use acoustic spectra of photoacoustic signals to quantify the absolute optical absorption. This method is self-calibrating and thus insensitive to variations in the optical fluence. Factors such as system bandwidth and acoustic attenuation can affect the quantification but can be canceled by dividing the acoustic spectra measured at two optical wavelengths. Using optical-resolution photoacoustic microscopy, we quantified the absolute optical absorption of black ink samples with various concentrations. We also quantified both the concentration and oxygen saturation of hemoglobin in a live mouse in absolute units.

  6. 3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

    PubMed

    Yio, M H N; Mac, M J; Wong, H S; Buenfeld, N R

    2015-05-01

    In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed.

  7. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  8. Implementation of PSF engineering in high-resolution 3D microscopy imaging with a LCoS (reflective) SLM

    NASA Astrophysics Data System (ADS)

    King, Sharon V.; Doblas, Ana; Patwary, Nurmohammed; Saavedra, Genaro; Martínez-Corral, Manuel; Preza, Chrysanthe

    2014-03-01

    Wavefront coding techniques are currently used to engineer unique point spread functions (PSFs) that enhance existing microscope modalities or create new ones. Previous work in this field demonstrated that simulated intensity PSFs encoded with a generalized cubic phase mask (GCPM) are invariant to spherical aberration or misfocus; dependent on parameter selection. Additional work demonstrated that simulated PSFs encoded with a squared cubic phase mask (SQUBIC) produce a depth invariant focal spot for application in confocal scanning microscopy. Implementation of PSF engineering theory with a liquid crystal on silicon (LCoS) spatial light modulator (SLM) enables validation of WFC phase mask designs and parameters by manipulating optical wavefront properties with a programmable diffractive element. To validate and investigate parameters of the GCPM and SQUBIC WFC masks, we implemented PSF engineering in an upright microscope modified with a dual camera port and a LCoS SLM. We present measured WFC PSFs and compare them to simulated PSFs through analysis of their effect on the microscope imaging system properties. Experimentally acquired PSFs show the same intensity distribution as simulation for the GCPM phase mask, the SQUBIC-mask and the well-known and characterized cubic-phase mask (CPM), first applied to high NA microscopy by Arnison et al.10, for extending depth of field. These measurements provide experimental validation of new WFC masks and demonstrate the use of the LCoS SLM as a WFC design tool. Although efficiency improvements are needed, this application of LCoS technology renders the microscope capable of switching among multiple WFC modes.

  9. Environmental monitoring of leaks using time-lapsed long electrode electrical resistivity

    NASA Astrophysics Data System (ADS)

    Rucker, Dale F.; Fink, James B.; Loke, Meng H.

    2011-08-01

    Highly industrialized areas pose challenges for surface electrical resistivity characterization due to metallic infrastructure. The infrastructure is typically more conductive than the desired targets and will mask the deeper subsurface information. The risk of this occurring may be minimized if steel-cased wells are used as long electrodes in the area near the target. We demonstrate a method of using long electrodes to electrically monitor a simulated leak from an underground storage tank with both synthetic examples and a field demonstration. Although the method of using long electrodes has been proposed by others, no time-lapse resistivity data have been collected, modeled, and analyzed within a nuclear waste tank farm environment. Therefore, the main objective of this work was to test whether the long electrode method using steel-cased wells can be employed to spatially and temporally track simulated leaks in a highly industrialized setting. A secondary objective was to apply a time-lapse regularization procedure in the inverse modeling code, similar to the 4D tomography approach by Kim et al. (2009), and to test the procedure's effect on the quality of the outcome regarding plume intensity and position. For the synthetic examples, a simple target of varying electrical properties was placed beneath different types of layers of low resistivity to simulate the effects of the infrastructure. Both surface and long electrodes were tested on the synthetic domain, and the test cases covered a variety of survey parameters including low and high electrode density, noise, array type, and the explicit location of the wells relative to the target. All data were processed in four dimensions, where the regularization procedure was applied in both the time and space domains. The synthetic test case showed that the long electrode resistivity method could detect relative changes in resistivity that was commensurate with the differing target properties. The surface electrodes

  10. Time-lapse capacitive resistivity imaging: a new technology concept for the monitoring of permafrost

    NASA Astrophysics Data System (ADS)

    Kuras, O.; Krautblatter, M.; Murton, J.; Haslam, E.; Wilkinson, P.; Meldrum, P.

    2011-12-01

    We have investigated and sought to prove a new technology concept for the non-invasive volumetric imaging and routine temporal monitoring of the thermal state of permafrost, a key indicator of global climate change. Capacitive Resistivity Imaging (CRI), a technique based upon low-frequency, capacitively-coupled measurements across permanently installed multi-sensor arrays is applied in order to emulate Electrical Resistivity Tomography (ERT) methodology, but without the need for galvanic contact on frozen soils or rocks. Recent work has shown that temperature-calibrated ERT using galvanic sensors is capable of imaging recession and re-advance of rock permafrost in response to the ambient temperature regime. However, our own laboratory experiments on rock samples under simulated permafrost conditions have equally demonstrated that galvanic electrodes experience large variations in contact resistances between sensors and the host material as the active layer freezes and thaws, leading to a rapid deterioration of data quality over time. As the presence of systematic but uncontrolled sensor noise will reduce the value of time-lapse ERT datasets for monitoring permafrost, the use of galvanic sensors will invariably impose practical limitations on field measurements. The capacitive methodology we are presenting here overcomes this problem and provides a roadmap for making stable resistance measurements with permanently installed sensors over time. We report on our experience with designing, building, testing and validating a functional prototype time-lapse CRI measurement system. The practical system architecture draws upon conceptual ideas incorporated in existing, field-scale CRI instrumentation designed by BGS; however, the use of dense capacitive sensor networks at the laboratory scale and the need for collecting tomographic imaging data across multiple sensors in an automated fashion required a novel technical approach. Our research has applied 4D CRI as well as

  11. Differences in morphogenesis of 3D cultured primary human osteoblasts under static and microfluidic growth conditions.

    PubMed

    Altmann, Brigitte; Löchner, Anne; Swain, Michael; Kohal, Ralf-Joachim; Giselbrecht, Stefan; Gottwald, Eric; Steinberg, Thorsten; Tomakidi, Pascal

    2014-03-01

    As information on osteoblast mechanosensitivity response to biomechanical cues in three-dimensional (3D) in vitro microenvironments is sparse, the present study compared morphogenesis of primary human alveolar bone osteoblasts (PHABO) under microchip-based 3D-static conditions, and 3D-fluid flow-mediated biomechanical stimulation in perfusion bioreactors. Discrimination of the respective microenvironment by differential morphogenesis was evident from fluid flow-induced PHABO reorganization into rotund bony microtissue, comprising more densely packed multicellular 3D-aggregates, while viability of microtissues was flow rate dependent. Time-lapse microscopy and simple modeling of biomechanical conditions revealed that physiologically relevant fluid flow-mediated PHABO stimulation was associated with formation of mulberry-like PHABO aggregates within the first 24 h. Differential extracellular matrix deposition patterns and gene expression modulation in PHABO aggregates at day 7 further indicates progressive osteoblast differentiation exclusively in perfusion culture-developed bony microtissues. The results of our study strongly suggest PHABO morphogenesis as discriminator of microenvironmental growth conditions, which in case of the microfluidic 3D microchip-bioreactor are substantiated by triggering in vitro bone microtissue formation concomitant with progressive osteoblastic differentiation. Such microtissue outcomes provide unique insight for mechanobiological studies in response to biomechanical fluid flow cues, and clinically appear promising for in vitro PHABO preconditioning, enabling innovative bone augmentation procedures.

  12. Fast history matching of time-lapse seismic and production data for high resolution models

    NASA Astrophysics Data System (ADS)

    Jimenez Arismendi, Eduardo Antonio

    Integrated reservoir modeling has become an important part of day-to-day decision analysis in oil and gas management practices. A very attractive and promising technology is the use of time-lapse or 4D seismic as an essential component in subsurface modeling. Today, 4D seismic is enabling oil companies to optimize production and increase recovery through monitoring fluid movements throughout the reservoir. 4D seismic advances are also being driven by an increased need by the petroleum engineering community to become more quantitative and accurate in our ability to monitor reservoir processes. Qualitative interpretations of time-lapse anomalies are being replaced by quantitative inversions of 4D seismic data to produce accurate maps of fluid saturations, pore pressure, temperature, among others. Within all steps involved in this subsurface modeling process, the most demanding one is integrating the geologic model with dynamic field data, including 4Dseismic when available. The validation of the geologic model with observed dynamic data is accomplished through a "history matching" (HM) process typically carried out with well-based measurements. Due to low resolution of production data, the validation process is severely limited in its reservoir areal coverage, compromising the quality of the model and any subsequent predictive exercise. This research will aim to provide a novel history matching approach that can use information from high-resolution seismic data to supplement the areally sparse production data. The proposed approach will utilize streamline-derived sensitivities as means of relating the forward model performance with the prior geologic model. The essential ideas underlying this approach are similar to those used for high-frequency approximations in seismic wave propagation. In both cases, this leads to solutions that are defined along "streamlines" (fluid flow), or "rays" (seismic wave propagation). Synthetic and field data examples will be used

  13. Morphokinetic behavior of euploid and aneuploid embryos analyzed by time-lapse in embryoscope

    PubMed Central

    Patel, Deven V.; Shah, Preeti B.; Kotdawala, Aditi P.; Herrero, Javier; Rubio, Irene; Banker, Manish R.

    2016-01-01

    BACKGROUND: Embryonic aneuploidy may result in miscarriage, implantation failure, or birth defects. Thus, it is clinically necessary to avoid the selection of aneuploid embryos during in vitro fertilization treatment. AIM: The aim of this study was to identify the morphokinetic differences by analyzing the development of euploid and aneuploid embryos using a time-lapse technology. We also checked the accuracy of a previously described model for selection of euploid embryos based on morphokinetics in our study population. MATERIALS AND METHODS: It is a retrospective study of 29 cycles undergoing preimplantation genetic screening from October 2013 to April 2015 at our center. Of 253 embryos, 167 suitable for biopsy embryos were analyzed for their chromosomal status using array-comparative genome hybridization (CGH). The morphokinetic behavior of these embryos was further analyzed in embryoscope using time-lapse technology. RESULTS: Among the analyzed embryos, 41 had normal and 126 had abnormal chromosome content. No significant difference in morphokinetics was found between euploid and aneuploid embryos. The percentage of embryos with blastulation was similar in the euploid (65.85%, 27/41) and aneuploid (60.31%, 76/126) embryos (P = 0.76). Although hard to define, majority of the chromosomal defects might be due to meiotic errors. On applying embryo selection model from Basile et al., embryos falling within optimal ranges for time to division to 5 cells (t5), time period of the third cell cycle (CC3), and time from 2 cell division to 5 cell division (t5-t2) exhibited greater proportion of normal embryos than those falling outside the optimal ranges (28.6%, 25.9%, and 26.7% vs. 17.5%, 20.8%, and 14.3%). CONCLUSION: Keeping a track of time interval between two stages can help us recognize aneuploid embryos at an earlier stage and prevent their selection of transfer. However, it cannot be used as a substitute for array CGH to select euploid embryos for transfer. PMID

  14. Geoelectrical time-lapse analysis for improved interpretation of data in a contaminated area

    NASA Astrophysics Data System (ADS)

    Chitea, Florina; Serban, Adrian; Ioane, Dumitru; Georgescu, Paul

    2014-05-01

    Non invasive geoelectrical studies are useful in the preliminary assessment of areas suspected to be contaminated but also in the investigation stage. Correctly adapted to the site specific situation, they are used to detect and investigate buried sources of pollution, to characterize the geology of the area, to detect the contaminated plume or to study the attenuation of pollution in case the appliance of an site-specific remediation techniques. Despite the improved technological acquisition techniques and the optimized inversion data algorithms, interpretation of geoelectrical data in still a challenging task, especially in a contaminated hydrogeological context. Beside the soil physical properties (composition, porosity, texture, etc.), moisture content and chemical composition of the pollutant are also influencing the measured parameter. Apparent electrical resistivity method was use in an area located near an Oil Refinery. Electrical measurements performed on profiles (transverse and along the direction of water flow -according to hydrological data) revealed the presence of contaminants by means of high resistivity anomalies. Using the same acquisition technique (Schlumberger array, same VES points, injection - AB - and voltage - MN - lines extension), measurements were repeated during time, along the same profiles. On the resulted electrical sections from 2006 to 2013, a dynamic situation regarding the pollution plume was observed. Time - lapse analysis, based on the calculation of resistivity differences between sets of data acquired along the same profile was applied, and data interpretation was made using the resulted sections. Significant variation between data sets (> 17% of apparent resistivity normalized differences) observed along the main profile were mainly ranging from the near surface (1.5 m) to an approximated depth (AB/2) of 10m. Using the time-lapse method, changes in the lateral and in depth extension of polluted areas could be observed and

  15. Three-dimensional scanning near field optical microscopy (3D-SNOM) imaging of random arrays of copper nanoparticles: implications for plasmonic solar cell enhancement.

    PubMed

    Ezugwu, Sabastine; Ye, Hanyang; Fanchini, Giovanni

    2015-01-07

    In order to investigate the suitability of random arrays of nanoparticles for plasmonic enhancement in the visible-near infrared range, we introduced three-dimensional scanning near-field optical microscopy (3D-SNOM) imaging as a useful technique to probe the intensity of near-field radiation scattered by random systems of nanoparticles at heights up to several hundred nm from their surface. We demonstrated our technique using random arrays of copper nanoparticles (Cu-NPs) at different particle diameter and concentration. Bright regions in the 3D-SNOM images, corresponding to constructive interference of forward-scattered plasmonic waves, were obtained at heights Δz ≥ 220 nm from the surface for random arrays of Cu-NPs of ∼ 60-100 nm in diameter. These heights are too large to use Cu-NPs in contact of the active layer for light harvesting in thin organic solar cells, which are typically no thicker than 200 nm. Using a 200 nm transparent spacer between the system of Cu-NPs and the solar cell active layer, we demonstrate that forward-scattered light can be conveyed in 200 nm thin film solar cells. This architecture increases the solar cell photoconversion efficiency by a factor of 3. Our 3D-SNOM technique is general enough to be suitable for a large number of other applications in nanoplasmonics.

  16. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

    PubMed

    Guilbert, Marie; Roig, Blandine; Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-02-23

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

  17. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model

    PubMed Central

    Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D.; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-01-01

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models. PMID:26885896

  18. Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography.

    PubMed

    Chen, Lingling; Alexandrov, Yuriy; Kumar, Sunil; Andrews, Natalie; Dallman, Margaret J; French, Paul M W; McGinty, James

    2015-04-01

    We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.

  19. Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy.

    PubMed

    Hughes, Louise; Borrett, Samantha; Towers, Katie; Starborg, Tobias; Vaughan, Sue

    2017-02-01

    The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle.

  20. Stroboscobic near-field scanning optical microscopy for 3D mapping of mode profiles of plasmonic nanostructures (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dana, Aykutlu; Ozgur, Erol; Torunoglu, Gamze

    2016-09-01

    We present a dynamic approach to scanning near field optical microscopy that extends the measurement technique to the third dimension, by strobing the illumination in sync with the cantilever oscillation. Nitrogen vacancy (NV) centers in nanodiamonds placed on cantilever tips are used as stable emitters for emission enhancement. Local field enhancement and modulation of optical density states are mapped in three dimensions based on fluorescence intensity and spectrum changes as the tip is scanned over plasmonic nanostructures. The excitation of NV centers is done using a total internal reflection setup. Using a digital phase locked loop to pulse the excitation in various tip sample separations, 2D slices of fluorescence enhancement can be recorded. Alternatively, a conventional SNOM tip can be used to selectively couple wideband excitation to the collection path, with subdiffraction resolution of 60 nm in x and y and 10 nm in z directions. The approach solves the problem of tip-sample separation stabilization over extended periods of measurement time, required to collect data resolved in emission wavelength and three spatial dimensions. The method can provide a unique way of accessing the three dimensional field and mode profiles of nanophotonics structures.

  1. Exceptionally Preserved Cambrian Trilobite Digestive System Revealed in 3D by Synchrotron-Radiation X-Ray Tomographic Microscopy

    PubMed Central

    Eriksson, Mats E.; Terfelt, Fredrik

    2012-01-01

    The Cambrian ‘Orsten’ fauna comprises exceptionally preserved and phosphatised microscopic arthropods. The external morphology of these fossils is well known, but their internal soft-tissue anatomy has remained virtually unknown. Here, we report the first non-biomineralised tissues from a juvenile polymerid trilobite, represented by digestive structures, glands, and connective strands harboured in a hypostome from the Swedish ‘Orsten’ fauna. Synchrotron-radiation X-ray tomographic microscopy enabled three-dimensional internal recordings at sub-micrometre resolution. The specimen provides the first unambiguous evidence for a J-shaped anterior gut and the presence of a crop with a constricted alimentary tract in the Trilobita. Moreover, the gut is Y-shaped in cross section, probably due to a collapsed lumen of that shape, another feature which has not previously been observed in trilobites. The combination of anatomical features suggests that the trilobite hypostome is functionally analogous to the labrum of euarthropods and that it was a sophisticated element closely integrated with the digestive system. This study also briefly addresses the preservational bias of the ‘Orsten’ fauna, particularly the near-absence of polymerid trilobites, and the taphonomy of the soft-tissue-harbouring hypostome. PMID:22558180

  2. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    SciTech Connect

    Fredrich, J.T.

    1999-02-10

    We present advances in the application of laser scanning confocal microscopy (LSCM) to image, reconstruct, and characterize statistically the microgeometry of porous geologic and engineering materials. We discuss technical and practical aspects of this imaging technique, including both its advantages and limitations. Confocal imaging can be used to optically section a material, with sub-micron resolution possible in the lateral and axial planes. The resultant volumetric image data, consisting of fluorescence intensities for typically {approximately}50 million voxels in XYZ space, can be used to reconstruct the three-dimensional structure of the two-phase medium. We present several examples of this application, including studying pore geometry in sandstone, characterizing brittle failure processes in low-porosity rock deformed under triaxial loading conditions in the laboratory, and analyzing the microstructure of porous ceramic insulations. We then describe approaches to extract statistical microgeometric descriptions from volumetric image data, and present results derived from confocal volumetric data sets. Finally, we develop the use of confocal image data to automatically generate a three-dimensional mesh for numerical pore-scale flow simulations.

  3. Exceptionally preserved Cambrian trilobite digestive system revealed in 3D by synchrotron-radiation X-ray tomographic microscopy.

    PubMed

    Eriksson, Mats E; Terfelt, Fredrik

    2012-01-01

    The Cambrian 'Orsten' fauna comprises exceptionally preserved and phosphatised microscopic arthropods. The external morphology of these fossils is well known, but their internal soft-tissue anatomy has remained virtually unknown. Here, we report the first non-biomineralised tissues from a juvenile polymerid trilobite, represented by digestive structures, glands, and connective strands harboured in a hypostome from the Swedish 'Orsten' fauna. Synchrotron-radiation X-ray tomographic microscopy enabled three-dimensional internal recordings at sub-micrometre resolution. The specimen provides the first unambiguous evidence for a J-shaped anterior gut and the presence of a crop with a constricted alimentary tract in the Trilobita. Moreover, the gut is Y-shaped in cross section, probably due to a collapsed lumen of that shape, another feature which has not previously been observed in trilobites. The combination of anatomical features suggests that the trilobite hypostome is functionally analogous to the labrum of euarthropods and that it was a sophisticated element closely integrated with the digestive system. This study also briefly addresses the preservational bias of the 'Orsten' fauna, particularly the near-absence of polymerid trilobites, and the taphonomy of the soft-tissue-harbouring hypostome.

  4. A Marked Poisson Process Driven Latent Shape Model for 3D Segmentation of Reflectance Confocal Microscopy Image Stacks of Human Skin.

    PubMed

    Ghanta, Sindhu; Jordan, Michael I; Kose, Kivanc; Brooks, Dana H; Rajadhyaksha, Milind; Dy, Jennifer G

    2016-10-05

    Segmenting objects of interest from 3D datasets is a common problem encountered in biological data. Small field of view and intrinsic biological variability combined with optically subtle changes of intensity, resolution and low contrast in images make the task of segmentation difficult, especially for microscopy of unstained living or freshly excised thick tissues. Incorporating shape information in addition to the appearance of the object of interest can often help improve segmentation performance. However, shapes of objects in tissue can be highly variable and design of a flexible shape model that encompasses these variations is challenging. To address such complex segmentation problems, we propose a unified probabilistic framework that can incorporate the uncertainty associated with complex shapes, variable appearance and unknown locations. The driving application which inspired the development of this framework is a biologically important segmentation problem: the task of automatically detecting and segmenting the dermal-epidermal junction (DEJ) in 3D reflectance confocal microscopy (RCM) images of human skin. RCM imaging allows noninvasive observation of cellular, nuclear and morphological detail. The DEJ is an important morphological feature as it is where disorder, disease and cancer usually start. Detecting the DEJ is challenging because it is a 2D surface in a 3D volume which has strong but highly variable number of irregularly spaced and variably shaped "peaks and valleys". In addition, RCM imaging resolution, contrast and intensity vary with depth. Thus a prior model needs to incorporate the intrinsic structure while allowing variability in essentially all its parameters. We propose a model which can incorporate objects of interest with complex shapes and variable appearance in an unsupervised setting by utilizing domain knowledge to build appropriate priors of the model. Our novel strategy to model this structure combines a spatial Poisson process with

  5. A Marked Poisson Process Driven Latent Shape Model for 3D Segmentation of Reflectance Confocal Microscopy Image Stacks of Human Skin.

    PubMed

    Ghanta, Sindhu; Jordan, Michael I; Kose, Kivanc; Brooks, Dana H; Rajadhyaksha, Milind; Dy, Jennifer G

    2017-01-01

    Segmenting objects of interest from 3D data sets is a common problem encountered in biological data. Small field of view and intrinsic biological variability combined with optically subtle changes of intensity, resolution, and low contrast in images make the task of segmentation difficult, especially for microscopy of unstained living or freshly excised thick tissues. Incorporating shape information in addition to the appearance of the object of interest can often help improve segmentation performance. However, the shapes of objects in tissue can be highly variable and design of a flexible shape model that encompasses these variations is challenging. To address such complex segmentation problems, we propose a unified probabilistic framework that can incorporate the uncertainty associated with complex shapes, variable appearance, and unknown locations. The driving application that inspired the development of this framework is a biologically important segmentation problem: the task of automatically detecting and segmenting the dermal-epidermal junction (DEJ) in 3D reflectance confocal microscopy (RCM) images of human skin. RCM imaging allows noninvasive observation of cellular, nuclear, and morphological detail. The DEJ is an important morphological feature as it is where disorder, disease, and cancer usually start. Detecting the DEJ is challenging, because it is a 2D surface in a 3D volume which has strong but highly variable number of irregularly spaced and variably shaped "peaks and valleys." In addition, RCM imaging resolution, contrast, and intensity vary with depth. Thus, a prior model needs to incorporate the intrinsic structure while allowing variability in essentially all its parameters. We propose a model which can incorporate objects of interest with complex shapes and variable appearance in an unsupervised setting by utilizing domain knowledge to build appropriate priors of the model. Our novel strategy to model this structure combines a spatial Poisson

  6. Time-Lapse Joint Inversion of Cross-Well DC Resistivity and Seismic Data: A Numerical Investigation

    EPA Science Inventory

    Time-lapse joint inversion of geophysical data is required to image the evolution of oil reservoirs during production and enhanced oil recovery, CO2 sequestration, geothermal fields during production, and to monitor the evolution of contaminant plumes. Joint inversion schemes red...

  7. The use of time lapse photography in an in vitro fertilization programme for better selection for embryo transfer.

    PubMed

    Kovačič, Borut; Hojnik, Nina; Vlaisavljević, Veljko

    2014-01-01

    The time lapse photography is not a new method for assessing the dynamics of early embryo development in vitro. It has been used many times in the past for studying cleavages and blastulation of embryos of various animal species. However, this technique became available for routine use in an human in vitro fertilization (IVF) programme only a couple years ago and it becomes more and more popular today. The new time lapse systems are using modified microscopes which are positioned within the incubators. The observation of embryos does not need the opening of incubators. By sequential photographing of each embryo separately with camera of low intensity illumination, more than 1400 pictures of embryo are made. All these pictures are collected together and transformed into a short movie with software. This system offers the observation of dynamics of embryo development. The studies, which have used a time lapse technique for studying embryo development, revealed that the timing between different events can be used for predicting its developmental potential. In this paper the advantages and drawbacks of time lapse photography is precisely described. An overview through the published papers analyzing the dynamics of human embryo development from the zygote toward blastocyst is done and new timing parameters for grading zygotes, early embryos and blastocysts are analyzed.

  8. 3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

    PubMed

    Khanmohammadi, M; Darkner, S; Nava, N; Nyengaard, J R; Wegener, G; Popoli, M; Sporring, J

    2017-01-01

    Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

  9. 3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

    PubMed

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  10. 3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

    PubMed Central

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M.

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  11. Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments.

    PubMed

    Gómez-Conde, Iván; Caetano, Susana S; Tadokoro, Carlos E; Olivieri, David N

    2015-09-01

    We describe a set of new algorithms and a software tool, StabiTissue, for stabilizing in vivo intravital microscopy images that suffer from soft-tissue background movement. Because these images lack predetermined anchors and are dominated by noise, we use a pixel weighted image alignment together with a correction for nonlinear tissue deformations. We call this correction a poor man׳s diffeomorphic map since it ascertains the nonlinear regions of the image without resorting to a full integral equation method. To determine the quality of the image stabilization, we developed an ensemble sampling method that quantifies the coincidence between image pairs from randomly distributed image regions. We obtain global stabilization alignment through an iterative constrained simulated annealing optimization procedure. To show the accuracy of our algorithm with existing software, we measured the misalignment error rate in datasets taken from two different organs and compared the results to a similar and popular open-source solution. Present open-source stabilization software tools perform poorly because they do not treat the specific needs of the IV-2pM datasets with soft-tissue deformation, speckle noise, full 5D inter- and intra-stack motion error correction, and undefined anchors. In contrast, the results of our tests demonstrate that our method is more immune to noise and provides better performance for datasets' possessing nonlinear tissue deformations. As a practical application of our software, we show how our stabilization improves cell tracking, where the presence of background movement would degrade track information. We also provide a qualitative comparison of our software with other open-source libraries/applications. Our software is freely available at the open source repository http://sourceforge.net/projects/stabitissue/.

  12. Mathematical analysis of endothelial sibling pair cell-cell interactions using time-lapse cinematography data.

    PubMed

    Brown, L M; Ryan, U S; Absher, M; Olazabal, B M

    1982-01-01

    The sibling pairs from two different endothelial cell cultures were analysed by time-lapse cinematography. It was shown that wounded and regular (low density seeded) cultures differed in the behaviour patterns of their siblings. The cultures differed most significantly in the minimum interdivision time (IDT) which was 27% lower for the wounded culture. In the wounded culture there was a greater correlation of IDT values between sibling pairs. IDT values recorded both for paired and for unpaired cells were shorter for the wounded than for the regular culture. The mean IDT for unpaired cells was longer than the mean IDT for paired cells in the regular culture. Thus paired cells in the regular culture, had shorter IDTs, but not as short as in the wounded culture. It was significant that in the wounded culture the first generation of siblings were very close (less than 150 microns apart) at division. Overall the behaviour differences between the two cultures resulted in a higher rate of increase in cell numbers, and thus faster repair, of the wounded monolayer.

  13. The impact of male age on embryo quality: a retrospective study using time-lapse imaging

    PubMed Central

    Rosário, Guilherme R. F.; Vidal, Diana S.; Silva, Adriana V.; Franco, Antônio C. C.

    2016-01-01

    Objective This study aimed to correlate male age with embryo morphokinetic parameters on D3 considering the timing and the exact moment of embryo cleavage. Methods Time-lapse imaging was used to produce an ideal cleavage curve for the embryos analyzed. The percentage of embryos under the curve was analyzed and correlated with male age. Results 32.6% of the embryos from patients aged 28-33 years were under the curve; 36.2% of the embryos from patients aged 34-39 years were under the curve; 41.3% of the embryos from patients aged 40-45 years were under the curve; and 26.3% of the embryos fro patients aged 46-57 years were under the curve. Conclusions a statistically non-significant decrease was observed in the percentage of embryos under the optimal cleavage curve on D3 in the group of men aged between 40 and 45 years. Further studies looking into embryos in the blastocyst stage (D5 or D6) are required. PMID:28050955

  14. ENVIRONMENTAL MONITORING OF LEAKS USING TIME LAPSED LONG ELECTRODE ELECTRICAL RESISTIVITY

    SciTech Connect

    RUCKER DF; FINK JB; LOKE MH; MYERS DA

    2009-11-05

    Highly industrialized areas pose significant challenges for surface based electrical resistivity characterization and monitoring due to the high degree of metallic infrastructure. The infrastructure is typically several orders of magnitude more conductive than the desired targets, preventing the geophysicist from obtaining a clear picture of the subsurface. These challenges may be minimized if steel-cased wells are used as long electrodes. We demonstrate a method of using long electrodes in a complex nuclear waste facility to monitor a simulated leak from an underground storage tank. The leak was simulated by injecting high conductivity fluid in a perforated well and the resistivity measurements were made before and after the leak test. The data were processed in four dimensions, where a regularization procedure was applied in both the time and space domains. The results showed a lowered resistivity feature develop south of the injection site. The time lapsed regularization parameter had a strong influence on the differences in inverted resistivity between the pre and post datasets, potentially making calibration of the results to specific hydrogeologic parameters difficult.

  15. Time-lapse imaging of human heart motion with switched array UWB radar.

    PubMed

    Brovoll, Sverre; Berger, Tor; Paichard, Yoann; Aardal, Øyvind; Lande, Tor Sverre; Hamran, Svein-Erik

    2014-10-01

    Radar systems for detection of human heartbeats have mostly been single-channel systems with limited spatial resolution. In this paper, a radar system for ultra-wideband (UWB) imaging of the human heart is presented. To make the radar waves penetrate the human tissue the antenna is placed very close to the body. The antenna is an array with eight elements, and an antenna switch system connects the radar to the individual elements in sequence to form an image. Successive images are used to build up time-lapse movies of the beating heart. Measurements on a human test subject are presented and the heart motion is estimated at different locations inside the body. The movies show rhythmic motion consistent with the beating heart, and the location and shape of the reflections correspond well with the expected response form the heart wall. The spatial dependent heart motion is compared to ECG recordings, and it is confirmed that heartbeat modulations are seen in the radar data. This work shows that radar imaging of the human heart may provide valuable information on the mechanical movement of the heart.

  16. Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation.

    PubMed

    Das, Amit; Gerlits, Oksana; Parks, Jerry M; Langan, Paul; Kovalevsky, Andrey; Heller, William T

    2015-12-01

    The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg(2+) binds first to the M1 site as a complex with ATP and is followed by Mg(2+) binding to the M2 site. Concurrently, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. Lastly, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.

  17. Direct prediction of spatially and temporally varying physical properties from time-lapse electrical resistance data

    NASA Astrophysics Data System (ADS)

    Hermans, Thomas; Oware, Erasmus; Caers, Jef

    2016-09-01

    Time-lapse applications of electrical methods have grown significantly over the last decade. However, the quantitative interpretation of tomograms in terms of physical properties, such as salinity, temperature or saturation, remains difficult. In many applications, geophysical models are transformed into hydrological models, but this transformation suffers from spatially and temporally varying resolution resulting from the regularization used by the deterministic inversion. In this study, we investigate a prediction-focused approach (PFA) to directly estimate subsurface physical properties with electrical resistance data, circumventing the need for classic tomographic inversions. First, we generate a prior set of resistance data and physical property forecast through hydrogeological and geophysical simulations mimicking the field experiment. We reduce the dimension of both the data and the forecast through principal component analysis in order to keep the most informative part of both sets in a reduced dimension space. Then, we apply canonical correlation analysis to explore the relationship between the data and the forecast in their reduced dimension space. If a linear relationship can be established, the posterior distribution of the forecast can be directly sampled using a Gaussian process regression where the field data scores are the conditioning data. In this paper, we demonstrate PFA for various physical property distributions. We also develop a framework to propagate the estimated noise level in the reduced dimension space. We validate the results by a Monte Carlo study on the posterior distribution and demonstrate that PFA yields accurate uncertainty for the cases studied.

  18. Time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain.

    PubMed

    Dong, Zhiqiang; Wagle, Mahendra; Guo, Su

    2011-04-06

    Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36 hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

  19. A time-lapse photography method for monitoring salmon (Oncorhynchus spp.) passage and abundance in streams

    PubMed Central

    Leacock, William B.; Eby, Lisa A.; Stanford, Jack A.

    2016-01-01

    Accurately estimating population sizes is often a critical component of fisheries research and management. Although there is a growing appreciation of the importance of small-scale salmon population dynamics to the stability of salmon stock-complexes, our understanding of these populations is constrained by a lack of efficient and cost-effective monitoring tools for streams. Weirs are expensive, labor intensive, and can disrupt natural fish movements. While conventional video systems avoid some of these shortcomings, they are expensive and require excessive amounts of labor to review footage for data collection. Here, we present a novel method for quantifying salmon in small streams (<15 m wide, <1 m deep) that uses both time-lapse photography and video in a model-based double sampling scheme. This method produces an escapement estimate nearly as accurate as a video-only approach, but with substantially less labor, money, and effort. It requires servicing only every 14 days, detects salmon 24 h/day, is inexpensive, and produces escapement estimates with confidence intervals. In addition to escapement estimation, we present a method for estimating in-stream salmon abundance across time, data needed by researchers interested in predator--prey interactions or nutrient subsidies. We combined daily salmon passage estimates with stream specific estimates of daily mortality developed using previously published data. To demonstrate proof of concept for these methods, we present results from two streams in southwest Kodiak Island, Alaska in which high densities of sockeye salmon spawn. PMID:27326378

  20. Landslide monitoring in southwestern China via time-lapse electrical resistivity tomography

    NASA Astrophysics Data System (ADS)

    Xu, Dong; Hu, Xiang-Yun; Shan, Chun-Ling; Li, Rui-Heng

    2016-03-01

    The dynamic monitoring of landslides in engineering geology has focused on the correlation among landslide stability, rainwater infiltration, and subsurface hydrogeology. However, the understanding of this complicated correlation is still poor and inadequate. Thus, in this study, we investigated a typical landslide in southwestern China via time-lapse electrical resistivity tomography (TLERT) in November 2013 and August 2014. We studied landslide mechanisms based on the spatiotemporal characteristics of surface water infiltration and flow within the landslide body. Combined with borehole data, inverted resistivity models accurately defined the interface between Quaternary sediments and bedrock. Preferential flow pathways attributed to fracture zones and fissures were also delineated. In addition, we found that surface water permeates through these pathways into the slipping mass and drains away as fissure water in the fractured bedrock, probably causing the weakly weathered layer to gradually soften and erode, eventually leading to a landslide. Clearly, TLERT dynamic monitoring can provide precursory information of critical sliding and can be used in landslide stability analysis and prediction.

  1. Imaging Rainfall Infiltration Processes with the Time-Lapse Electrical Resistivity Imaging Method

    NASA Astrophysics Data System (ADS)

    Zhang, Gang; Zhang, Gui-Bin; Chen, Chien-chih; Chang, Ping-Yu; Wang, Tzu-Pin; Yen, Horng-Yuan; Dong, Jia-Jyun; Ni, Chuen-Fa; Chen, Su-Chin; Chen, Chao-Wei; Jia, Zheng-yuan

    2016-06-01

    Electrical Resistivity Imaging (ERI) was carried out continuously for 10 days to map the subsurface resistivity distribution along a potentially hazardous hillslope at the Jieshou Junior High School in Taoyuan, Taiwan. The reliability of the inverted resistivity structures down to about 25 m depth was examined with synthetic modeling using the same electrode arrangements installed on land surface as in field surveys, together with a DOI (depth-of-investigation) index calculated from the ERI data. The subsurface resistivity distribution is consistent with results from well logging. These ERI recordings were taken daily and provided highly resolved imagery of the resistivity distribution underground and illustrated the dynamical fluid-flow behavior due to heavy rainfall infiltration. Using Archie's law, the resistivity distribution was transformed into a map of relative water saturation (RWS), which is strongly correlated with the rainfall infiltration process. We then found that the averaged RWS is significantly correlated with daily precipitation. Our observations indicate that time-lapse ERI is effective in monitoring subterraneous rainfall infiltration; moreover, the preferential flow paths can be delineated according to the changes in averaged RWS derived from the ERI data.

  2. Non-rigid estimation of cell motion in calcium time-lapse images

    NASA Astrophysics Data System (ADS)

    Hachi, Siham; Lucumi Moreno, Edinson; Desmet, An-Sofie; Vanden Berghe, Pieter; Fleming, Ronan M. T.

    2016-03-01

    Calcium imaging is a widely used technique in neuroscience permitting the simultaneous monitoring of electro- physiological activity of hundreds of neurons at single cell resolution. Identification of neuronal activity requires rapid and reliable image analysis techniques, especially when neurons fire and move simultaneously over time. Traditionally, image segmentation is performed to extract individual neurons in the first frame of a calcium sequence. Thereafter, the mean intensity is calculated from the same region of interest in each frame to infer calcium signals. However, when cells move, deform and fire, this segmentation on its own generates artefacts and therefore biased neuronal activity. Therefore, there is a pressing need to develop a more efficient cell tracking technique. We hereby present a novel vision-based cell tracking scheme using a thin-plate spline deformable model. The thin-plate spline warping is based on control points detected using the Fast from Accelerated Segment Test descriptor and tracked using the Lucas-Kanade optical flow. Our method is able to track neurons in calcium time-series, even when there are large changes in intensity, such as during a firing event. The robustness and efficiency of the proposed approach is validated on real calcium time-lapse images of a neuronal population.

  3. Time-Lapse Dynamics of the Mouse Oocyte Chromatin Organisation during Meiotic Resumption

    PubMed Central

    Redi, Carlo Alberto; Zuccotti, Maurizio

    2014-01-01

    In the mammalian oocyte, distinct patterns of centromeres and pericentromeric heterochromatin localisation correlate with the gamete's developmental competence. Mouse antral oocytes display two main types of chromatin organisation: SN oocytes, with a ring of Hoechst-positive chromatin surrounding the nucleolus, and NSN oocytes lacking this ring. When matured to MII and fertilised, only SN oocytes develop beyond the 2-cell, and reach full term. To give detailed information on the dynamics of the SN or NSN chromatin during meiosis resumption, we performed a 9 hr time-lapse observation. The main significant differences recorded are: (1) reduction of the nuclear area only in SN oocytes; (2) ~17 min delay of GVBD in NSN oocytes; (3) chromatin condensation, after GVBD, in SN oocytes; (4) formation of 4-5 CHCs in SN oocytes; (5) increase of the perivitelline space, ~57 min later in NSN oocytes; (6) formation of a rosette-like disposition of CHCs, ~84 min later in SN oocytes; (7) appearance of the MI plate ~40 min later in NSN oocytes. Overall, we described a pathway of transition from the GV to the MII stage that is punctuated of discrete recordable events showing their specificity and occurring with different time kinetics in the two types of oocytes. PMID:24864231

  4. Thermal erosion of a permafrost coastline: Improving process-based models using time-lapse photography

    USGS Publications Warehouse

    Wobus, C.; Anderson, R.; Overeem, I.; Matell, N.; Clow, G.; Urban, F.

    2011-01-01

    Coastal erosion rates locally exceeding 30 m y-1 have been documented along Alaska's Beaufort Sea coastline, and a number of studies suggest that these erosion rates have accelerated as a result of climate change. However, a lack of direct observational evidence has limited our progress in quantifying the specific processes that connect climate change to coastal erosion rates in the Arctic. In particular, while longer ice-free periods are likely to lead to both warmer surface waters and longer fetch, the relative roles of thermal and mechanical (wave) erosion in driving coastal retreat have not been comprehensively quantified. We focus on a permafrost coastline in the northern National Petroleum Reserve-Alaska (NPR-A), where coastal erosion rates have averaged 10-15 m y-1 over two years of direct monitoring. We take advantage of these extraordinary rates of coastal erosion to observe and quantify coastal erosion directly via time-lapse photography in combination with meteorological observations. Our observations indicate that the erosion of these bluffs is largely thermally driven, but that surface winds play a crucial role in exposing the frozen bluffs to the radiatively warmed seawater that drives melting of interstitial ice. To first order, erosion in this setting can be modeled using formulations developed to describe iceberg deterioration in the open ocean. These simple models provide a conceptual framework for evaluating how climate-induced changes in thermal and wave energy might influence future erosion rates in this setting.

  5. Estimating the onset of time-lapse changes: A robust basis for reservoir monitoring and characterization

    NASA Astrophysics Data System (ADS)

    Vasco, D. W.

    2013-12-01

    Time-lapse monitoring is useful for imaging flow related changes in geophysical attributes. However, it can be difficult to relate changes in geophysical observations to changes in saturation and pressure in a reservoir. As an alternative approach for reservoir monitoring and characterization, we introduce the idea of an onset time, the time at which measured quantities, such as seismic travel times or reflection amplitudes, begin to deviate from their background values. We illustrate the idea of an onset time and demonstrate its utility through the consideration of travel times recorded by the Continuous Active Source Seismic Monitoring (CASSM) system at the Frio pilot site near Houston, Texas. The system, which transmits an elastic wave every 15 minutes, is used to monitor the movement of carbon dioxide injected into a permeable sand formation. From these data we can estimate the onset of changes in seismic travel times to six receivers in an adjacent borehole. Numerical simulation and synthetic tests indicate that the onset times are not very sensitive to the method used to compute the effective fluid bulk modulus and, correspondingly, the seismic compressional velocity. Rather, the onset times are strongly influenced by saturation changes within the formation, specifically by the break-through time of the injected fluid phase. By means of an iterative inversion algorithm we use the onset times to estimate permeability variations between the boreholes at the Frio pilot site.

  6. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

    PubMed Central

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun’ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  7. Investigating motion blur and temporal aliasing from time-lapse electrical resistivity

    NASA Astrophysics Data System (ADS)

    Rucker, Dale

    2014-12-01

    Geophysical monitoring through time-lapsed resistivity imaging is investigated to determine detrimental effects resulting from temporal smear. Temporal smear can be divided into motion blur and temporal aliasing, with motion blur attributed to an extended sample integration time relative to the velocity of a moving target, thus giving rise to reproduced targets that are distorted versions of the real target shape. Aliasing results from undersampling across time and may give a discontinuous movement. The degree to which each aspect of smear affects target properties described by spatial moment analysis depends on the spatial resolution of the imaging method and the degree to which temporal degradation is applied. For synthetic models with relatively high spatial resolution, aliasing effects were slight except in cases where the minimal number of snapshots was acquired to understand the end state condition of the target. Motion blur, on the other hand, had progressive detrimental effects with each level of additional smearing. For field data acquired during subsurface injection with a lower resolution array, the damaging effects from motion blur and temporal aliasing were equivalent. Both aspects showed progressive degeneration of spatial moments with each level of degradation. To combat this problem in the short term, it is recommended to acquire resistivity data as rapidly as possible and sacrifice some spatial resolution to enhance temporal resolution. In the future, there may be methods adopted from motion photography to deblur target motion by using the point spread function. Aliasing, however, can only be solved through continuous sampling.

  8. Cellular dynamics during early barley pollen embryogenesis revealed by time-lapse imaging

    PubMed Central

    Daghma, Diaa Eldin S.; Hensel, Goetz; Rutten, Twan; Melzer, Michael; Kumlehn, Jochen

    2014-01-01

    Plants display a remarkable capacity for cellular totipotency. An intriguing and useful example is that immature pollen cultured in vitro can pass through embryogenic development to form haploid or doubled haploid plants. However, a lack of understanding the initial mechanisms of pollen embryogenesis hampers the improvement and more effective and widespread employment of haploid technology in plant research and breeding. To investigate the cellular dynamics during the onset of pollen embryogenesis, we used time-lapse imaging along with transgenic barley expressing nuclear localized Green Fluorescent Protein. The results enabled us to identify nine distinct embryogenic and non-embryogenic types of pollen response to the culture conditions. Cell proliferation in embryogenic pollen normally started via a first symmetric mitosis (54.3% of pollen observed) and only rarely did so via asymmetric pollen mitosis I (4.3% of pollen observed). In the latter case, proliferation generally originated from the vegetative-like cell, albeit the division of the generative-like cell was observed in few types of pollen. Under the culture conditions used, fusion of cell nuclei was the only mechanism of genome duplication observed. PMID:25538715

  9. Time-lapse characterization of hydrothermal seawater and microbial interactions with basaltic tephra at Surtsey Volcano

    NASA Astrophysics Data System (ADS)

    Jackson, M. D.; Gudmundsson, M. T.; Bach, W.; Cappelletti, P.; Coleman, N. J.; Ivarsson, M.; Jónasson, K.; Jørgensen, S. L.; Marteinsson, V.; McPhie, J.; Moore, J. G.; Nielson, D.; Rhodes, J. M.; Rispoli, C.; Schiffman, P.; Stefánsson, A.; Türke, A.; Vanorio, T.; Weisenberger, T. B.; White, J. D. L.; Zierenberg, R.; Zimanowski, B.

    2015-12-01

    A new International Continental Drilling Program (ICDP) project will drill through the 50-year-old edifice of Surtsey Volcano, the youngest of the Vestmannaeyjar Islands along the south coast of Iceland, to perform interdisciplinary time-lapse investigations of hydrothermal and microbial interactions with basaltic tephra. The volcano, created in 1963-1967 by submarine and subaerial basaltic eruptions, was first drilled in 1979. In October 2014, a workshop funded by the ICDP convened 24 scientists from 10 countries for 3 and a half days on Heimaey Island to develop scientific objectives, site the drill holes, and organize logistical support. Representatives of the Surtsey Research Society and Environment Agency of Iceland also participated. Scientific themes focus on further determinations of the structure and eruptive processes of the type locality of Surtseyan volcanism, descriptions of changes in fluid geochemistry and microbial colonization of the subterrestrial deposits since drilling 35 years ago, and monitoring the evolution of hydrothermal and biological processes within the tephra deposits far into the future through the installation of a Surtsey subsurface observatory. The tephra deposits provide a geologic analog for developing specialty concretes with pyroclastic rock and evaluating their long-term performance under diverse hydrothermal conditions. Abstracts of research projects are posted at http://surtsey.icdp-online.org.

  10. 3D digital image processing for biofilm quantification from confocal laser scanning microscopy: Multidimensional statistical analysis of biofilm modeling

    NASA Astrophysics Data System (ADS)

    Zielinski, Jerzy S.

    The dramatic increase in number and volume of digital images produced in medical diagnostics, and the escalating demand for rapid access to these relevant medical data, along with the need for interpretation and retrieval has become of paramount importance to a modern healthcare system. Therefore, there is an ever growing need for processed, interpreted and saved images of various types. Due to the high cost and unreliability of human-dependent image analysis, it is necessary to develop an automated method for feature extraction, using sophisticated mathematical algorithms and reasoning. This work is focused on digital image signal processing of biological and biomedical data in one- two- and three-dimensional space. Methods and algorithms presented in this work were used to acquire data from genomic sequences, breast cancer, and biofilm images. One-dimensional analysis was applied to DNA sequences which were presented as a non-stationary sequence and modeled by a time-dependent autoregressive moving average (TD-ARMA) model. Two-dimensional analyses used 2D-ARMA model and applied it to detect breast cancer from x-ray mammograms or ultrasound images. Three-dimensional detection and classification techniques were applied to biofilm images acquired using confocal laser scanning microscopy. Modern medical images are geometrically arranged arrays of data. The broadening scope of imaging as a way to organize our observations of the biophysical world has led to a dramatic increase in our ability to apply new processing techniques and to combine multiple channels of data into sophisticated and complex mathematical models of physiological function and dysfunction. With explosion of the amount of data produced in a field of biomedicine, it is crucial to be able to construct accurate mathematical models of the data at hand. Two main purposes of signal modeling are: data size conservation and parameter extraction. Specifically, in biomedical imaging we have four key problems

  11. Monitoring channel head erosion processes in response to an artificially induced abrupt base level change using time-lapse photography

    NASA Astrophysics Data System (ADS)

    Nichols, M. H.; Nearing, M.; Hernandez, M.; Polyakov, V. O.

    2016-07-01

    Gullies that terminate at a vertical-wall are ubiquitous throughout arid and semiarid regions. Multi-year assessments of gully evolution and headcut advance are typically accomplished using traditional ground surveys and aerial photographs, with much recent research focused on integrating data collected at very high spatial resolutions using new techniques such as aerial surveys with blimps or kites and ground surveys with LiDar scanners. However, knowledge of specific processes that drive headcut advance is limited due to inadequate observation and documentation of flash floods and subsequent erosion that can occur at temporal resolutions not captured through repeat surveys. This paper presents a method for using very-high temporal resolution ground-based time-lapse photography to capture short-duration flash floods and gully head evolution in response. In 2004, a base level controlling concrete weir was removed from the outlet of a 1.29 ha semiarid headwater drainage on the Walnut Gulch Experimental Watershed in southeastern Arizona, USA. During the ten year period from 2004 to 2014 the headcut migrated upchannel a total of 14.5 m reducing the contributing area at the headwall by 9.5%. Beginning in July 2012, time-lapse photography was employed to observe event scale channel evolution dynamics. The most frequent erosion processes observed during three seasons of time-lapse photography were plunge pool erosion and mass wasting through sidewall or channel headwall slumping that occurred during summer months. Geomorphic change during the ten year period was dominated by a single piping event in August 2014 that advanced the channel head 7.4 m (51% of the overall advance) and removed 11.3 m3 of sediment. High temporal resolution time-lapse photography was critical for identifying subsurface erosion processes, in the absence of time-lapse images piping would not have been identified as an erosion mechanism responsible for advancing the gully headwall at this site.

  12. Analysis of one year time-lapse electrical data to unravel hydrological processes acting on a clayey landslide

    NASA Astrophysics Data System (ADS)

    Gance, J.; Sailhac, P.; Malet, J.; Supper, R.; Jochum, B.; Ottowitz, D.; Grandjean, G.

    2013-12-01

    Movements of water in the topsoil (infiltration, run-off and evaporation) influence changes in slope stability which is the main controlling factor of landslide triggering (e.g. van Asch et al., 1999). Among the petrophysical parameters that can provide time-lapse sections of the topsoil, we consider the electrical conductivity for its sensitivity to soil water contents. Based on recent works which showed the possibility of monitoring the hydrological response of a clay-shale slope to a controlled rainfall experiment (Travelletti et al., 2012), we installed a permanent electrical monitoring experiment at the Super-Sauze landslide for long-term monitoring (one year) of natural meteorological events. We used the GEOMON4D resistivimeter, developed by the Austrian Geological Survey (Vienna, Austria) for experiments needing high rate of data acquisition, records of full signal samples for noise detection, remote controlled management and automatic data transfer (Supper et al., 2002, 2003 & 2004). The electrode positions varying with time, we installed two terrestrial optical cameras to characterize the changes in dipole geometry. Several hydrological sensors were installed along the profile to measure soil temperature, water temperature and conductivity, ground water level and soil humidity in the vadose zone. The main challenge is the processing of ca. 4.2 million of electrical resistivity data. In this difficult context, the potential factors influencing electrical resistivity with time without modification of soil saturation are the relative changes in the dipole geometry (linked to the displacement of the electrodes), changes in soil and water temperature, change in material porosity due to compaction/dilatation caused by the landslide movement. Therefore, before any inversion of data, we verify the presence of possible 3D effects, and assess the measurement accuracy and uncertainty. An apparent resistivity variation threshold, from which a modification of the

  13. Three-dimensional scanning near field optical microscopy (3D-SNOM) imaging of random arrays of copper nanoparticles: implications for plasmonic solar cell enhancement

    NASA Astrophysics Data System (ADS)

    Ezugwu, Sabastine; Ye, Hanyang; Fanchini, Giovanni

    2014-11-01

    In order to investigate the suitability of random arrays of nanoparticles for plasmonic enhancement in the visible-near infrared range, we introduced three-dimensional scanning near-field optical microscopy (3D-SNOM) imaging as a useful technique to probe the intensity of near-field radiation scattered by random systems of nanoparticles at heights up to several hundred nm from their surface. We demonstrated our technique using random arrays of copper nanoparticles (Cu-NPs) at different particle diameter and concentration. Bright regions in the 3D-SNOM images, corresponding to constructive interference of forward-scattered plasmonic waves, were obtained at heights Δz >= 220 nm from the surface for random arrays of Cu-NPs of ~60-100 nm in diameter. These heights are too large to use Cu-NPs in contact of the active layer for light harvesting in thin organic solar cells, which are typically no thicker than 200 nm. Using a 200 nm transparent spacer between the system of Cu-NPs and the solar cell active layer, we demonstrate that forward-scattered light can be conveyed in 200 nm thin film solar cells. This architecture increases the solar cell photoconversion efficiency by a factor of 3. Our 3D-SNOM technique is general enough to be suitable for a large number of other applications in nanoplasmonics.In order to investigate the suitability of random arrays of nanoparticles for plasmonic enhancement in the visible-near infrared range, we introduced three-dimensional scanning near-field optical microscopy (3D-SNOM) imaging as a useful technique to probe the intensity of near-field radiation scattered by random systems of nanoparticles at heights up to several hundred nm from their surface. We demonstrated our technique using random arrays of copper nanoparticles (Cu-NPs) at different particle diameter and concentration. Bright regions in the 3D-SNOM images, corresponding to constructive interference of forward-scattered plasmonic waves, were obtained at heights Δz >= 220

  14. Automatic detection and analysis of cell motility in phase-contrast time-lapse images using a combination of maximally stable extremal regions and Kalman filter approaches.

    PubMed

    Kaakinen, M; Huttunen, S; Paavolainen, L; Marjomäki, V; Heikkilä, J; Eklund, L

    2014-01-01

    Phase-contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase-contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase-contrast images in time-lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time-lapse movies, the MSER-based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase-contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time-consuming large-scale dynamical analysis of cultured cells.

  15. Overland flow dynamics through visual observation using time-lapse photographs

    NASA Astrophysics Data System (ADS)

    Silasari, Rasmiaditya; Blöschl, Günter

    2016-04-01

    Overland flow process on agricultural land is important to be investigated as it affects the stream discharge and water quality assessment. During rainfall events the formation of overland flow may happen through different processes (i.e. Hortonian or saturation excess overland flow) based on the governing soil hydraulic parameters (i.e. soil infiltration rate, soil water capacity). The dynamics of the soil water state and the processes will affect the surface runoff response which can be analyzed visually by observing the saturation patterns with a camera. Although visual observation was proven useful in laboratory experiments, the technique is not yet assessed for natural rainfall events. The aim of this work is to explore the use of time-lapse photographs of naturally occurring-saturation patterns in understanding the threshold processes of overland flow generation. The image processing produces orthographic projection of the saturation patterns which will be used to assess the dynamics of overland flow formation in relation with soil moisture state and rainfall magnitude. The camera observation was performed at Hydrological Open Air Laboratory (HOAL) catchment at Petzenkirchen, Lower Austria. The catchment covers an area of 66 ha dominated with agricultural land (87%). The mean annual precipitation and mean annual flow at catchment outlet are 750 mm and 4 l/s, respectively. The camera was set to observe the overland flow along a thalweg on an arable field which was drained in 1950s and has advantages of: (1) representing agricultural land as the dominant part of the catchment, (2) adjacent to the stream with clear visibility (no obstructing objects, such as trees), (3) drained area provides extra cases in understanding the response of tile drain outflow to overland flow formation and vice versa, and (4) in the vicinity of TDT soil moisture stations. The camera takes a picture with 1280 x 720 pixels resolution every minute and sends it directly in a PC via fiber

  16. Time Lapse Storey Building Early Monitoring Based on Rapid Seismic Response Analysis in Indonesia

    NASA Astrophysics Data System (ADS)

    Julius, A. M.

    2015-12-01

    Within the last decade, advances in the acquisition, processing and transmission of data from seismic monitoring has contributed to the growth in the number structures instrumented with such systems. An equally important factor for such growth can be attributed to the demands by stakeholders to find rapid answers to important questions related to the functionality or state of "health" of structures during and immediately of a seismic events. Consequently, this study aims to monitor the storey building based on seismic response i. e. earthquake and tremor analysis at short time lapse using accelerographs data. This study used one of storey building (X) in Jakarta city that suffered the effects of Kebumen earthquake January 25th 2014, Pandeglang earthquake July 9th 2014, and Lebak earthquake November 8th 2014. Tremors used in this study are tremors after the three following earthquakes. Data processing used to determine peak ground acceleration (PGA), peak ground velocity (PGV), peak ground displacement (PGD), spectral acceleration (SA), spectral velocity (SV), spectral displacement (SD), A/V ratio, acceleration amplification and effective duration (te). Then determine the natural frequency (f0) and peak of H/V ratio using H/V ratio method. The earthquakes data processing result shows the value of peak ground motion, spectrum response, A/V ratio and acceleration amplification increases with height, while the value of the effective duration decreases. Then, tremors data processing result one month after each earthquakes shows the natural frequency of building in constant value. Increasing of peak ground motion, spectrum response, A/V ratio, acceleration amplification, then decrease of effective duration following the increase of building floors shows that the building construction supports the increasing of shaking and strongly influenced by local site effect. The constant value of building natural frequency shows the building still in good performance. This

  17. Time Lapse Gravity and Seismic Monitoring of CO2 Injection at the West Hastings Field, Texas

    NASA Astrophysics Data System (ADS)

    Ferguson, J. F.; Richards, T.; Klopping, F.; MacQueen, J.; Hosseini, S. A.

    2015-12-01

    Time lapse or 4D gravity and seismic reflection surveys are being conducted at the West Hastings Field near Houston, Texas to monitor the progress of CO2 injection. This Department of Energy supported CO2 sequestration experiment is conducted in conjunction with a Denbury Onshore, LLC tertiary recovery project. The reservoir is at a depth of 1.8 km in the Oligocene Frio sands and has been produced since the 1930s. Goals are an accounting and mapping of the injected CO2 and to determine if migration occurs along intra-reservoir faults. An integrated interpretation of the geophysical surveys will be made together with well logs and engineering data. Gravity monitoring of water versus gas replacement has been very successful, but liquid phase CO2 monitoring is problematic due to the smaller density contrast with respect to oil and water. This reservoir has a small volume to depth ratio and hence only a small gravity difference signal is expected on the surface. New borehole gravity technology introduced by Micro-g-Lacoste can make gravity measurements at near reservoir depths with a much higher signal to noise ratio. This method has been successfully evaluated on a simulation of the Hastings project. Field operations have been conducted for repeated surface and borehole gravity surveys beginning in 2013. The surface survey of 95 stations covers an area of 3 by 5 km and 22 borehole gravity logs are run in the interval above the Frio formation. 4D seismic reflection surveys are being made at 6 month intervals on the surface and in 3 VSP wells. CO2 injection into the targeted portion of the reservoir only began in early 2015 and monitoring will continue into 2017. To date only the baseline reservoir conditions have been assessed. The overall success of the gravity monitoring will not be determined until 2017.

  18. A Local Index of Cloud Immersion in Tropical Forests Using Time-Lapse Photography

    NASA Astrophysics Data System (ADS)

    Bassiouni, M.; Scholl, M. A.

    2015-12-01

    Data on the frequency, duration and elevation of cloud immersion is essential to improve estimates of cloud water deposition in water budgets in cloud forests. Here, we present a methodology to detect local cloud immersion in remote tropical forests using time-lapse photography. A simple approach is developed to detect cloudy conditions in photographs within the canopy where image depth during clear conditions may be less than 10 meters and moving leaves and branches and changes in lighting are unpredictable. A primary innovation of this study is that cloudiness is determined from images without using a reference clear image and without minimal threshold value determination or human judgment for calibration. Five sites ranging from 600 to 1000 meters elevation along a ridge in the Luquillo Critical Zone Observatory, Puerto Rico were each equipped with a trail camera programmed to take an image every 30 minutes since March 2014. Images were classified using four selected cloud-sensitive image characteristics (SCICs) computed for small image regions: contrast, the coefficient of variation and the entropy of the luminance of each image pixel, and image colorfulness. K-means clustering provided reasonable results to discriminate cloudy from clear conditions. Preliminary results indicate that 79-94% (daytime) and 85-93% (nighttime) of validation images were classified accurately at one open and two closed canopy sites. The euclidian distances between SCICs vectors of images during cloudy conditions and the SCICs vector of the centroid of the cluster of clear images show potential to quantify cloud density in addition to immersion. The classification method will be applied to determine spatial and temporal patterns of cloud immersion in the study area. The presented approach offers promising applications to increase observations of low-lying clouds at remote mountain sites where standard instruments to measure visibility and cloud base may not be practical.

  19. Using electromagnetic conductivity imaging to generate time-lapse soil moisture estimates.

    NASA Astrophysics Data System (ADS)

    Huang, Jingyi; Scuderio, Elia; Corwin, Dennis; Triantafilis, John

    2015-04-01

    Irrigated agriculture is crucial to the agricultural productivity of the Moreno valley. To maintain profitability, more will need to be done by irrigators with less water, owing to competing demands from rapidly expanding urbanisation in southern California. In this regard, irrigators need to understand the spatial and temporal variation of soil moisture to discern inefficiencies. However, soil moisture is difficult to measure and monitor, unless a large bank of soil sensors are installed and at various depths in the profile. In order to value add to the limited amount of information, geophysical techniques, such as direct current resisivity (DCR) arrays are used to develop electrical resistivity images (ERI). Whilst successful the approach is time consuming and labour intensive. In this research we describe how equivalent data can be collected using a proximal sensing electromagnetic (EM) induction instrument (i.e. DUALEM-421) and inversion software (EM4Soil) to generate EM conductivity images (EMCI). Figure 1 shows the EMCI generated from DUALEM-421 data acquired at various days of a time-lapse experiment and including; day a) 0, b) 1, c) 2, d) 3, e) 5, f) 7 and g) 11. We calibrate the estimates of true electrical conductivity (sigma - mS/m) with volumetric moisture content and show with good accuracy the spatial and temporal variation of soil moisture status and over 12 day period. The results show clearly that the pivot sprinkler irrigation system is effective at providing sufficient amounts of water to the top 0.5 m of a Lucerne crop (i.e. red shaded areas of high sigma). However, in some places faulty sprinklers are evident owing to the lack of wetting (i.e. blue shaded areas of low sigma). In addition, and over time, our approach shows clearly the effect the Lucerne crop has in drying the soil profile and using the soil moisture.

  20. ENVIRONMENTAL MONITORING OF LEAKS USING TIME LAPSED LONG ELECTRODE ELECTRICAL RESISTIVITY

    SciTech Connect

    MYERS DA; RUCKER DF; FINK JB; LOKE MH

    2009-12-16

    Highly industrialized areas pose challenges for surface electrical resistivity characterization due to metallic infrastructure. The infrastructure is typically more conductive than the desired targets and will mask the deeper subsurface information. These challenges may be minimized if steel-cased wells are used as long electrodes in the area near the target. We demonstrate a method of using long electrodes to electrically monitor a simulated leak from an underground storage tank with both synthetic examples and a field demonstration. The synthetic examples place a simple target of varying electrical properties beneath a very low resistivity layer. The layer is meant to replicate the effects of infrastructure. Both surface and long electrodes are tested on the synthetic domain. The leak demonstration for the field experiment is simulated by injecting a high conductivity fluid in a perforated well within the S tank farm at Hanford, and the resistivity measurements are made before and after the leak test. All data are processed in four dimensions, where a regularization procedure is applied in both the time and space domains. The synthetic test case shows that the long electrode ERM could detect relative changes in resistivity that are commensurate with the differing target properties. The surface electrodes, on the other hand, had a more difficult time matching the original target's footprint. The field results shows a lowered resistivity feature develop south of the injection site after cessation of the injections. The time lapsed regularization parameter has a strong influence on the differences in inverted resistivity between the pre and post injection datasets, but the interpretation of the target is consistent across all values of the parameter. The long electrode ERM method may provide a tool for near real-time monitoring of leaking underground storage tanks.

  1. Assessing spatial and temporal snowpack evolution and melt with time-lapse photography

    NASA Astrophysics Data System (ADS)

    Bush, C. E.; Ewers, B. E.; Beverly, D.; Speckman, H. N.; Hyde, K.; Ohara, N.

    2015-12-01

    Snowpack supplies and stores water for many ecosystems of the greater Rocky Mountain region. In Wyoming the snowpack supplies water to 18 states east and west of the Continental Divide. The spatial variability in physical and biological processes creates a heterogeneous pattern of snow evolution. Understanding these processes within individual plots and throughout the entire watershed increases the predictive power of snow distribution, melt rates and contribution to streamflow. However, on site sampling of snow can be an expensive and arduous process. The objective of this experiment was to quantify spatial and temporal patterns of snowpack evolution and melt rates while minimizing perturbations to snowpack through the use of time-lapse photography via trail cameras. Field cameras were assessed as a method to quantify snow depths throughout the 120 ha No Name watershed at approximately 3000 m elevation in central Wyoming. RGB trail cameras were installed at three systematically chosen sites within the watershed to correlate physical and biological drivers of snow distribution. Five stakes were placed in each site in heterogeneous spots that remained in the frame of the camera. Stakes were divided into five centimeter increments, alternating black and white bars, with red bars denoting each half meter. Images were then taken at two-hour intervals over a period of three-months and analyzed with the ImageJ program. Snowpack distributions, as well as melt rates, were variable at both the plot and watershed scales. Meteorological and physical drivers, primarily topography and radiation, accounted for the greatest variability when comparing among plot across the watershed; however, LAI and soil and air temperature were the most significant drivers within plots. Snow-melt rate increased as soils and course woody debris became exposed increasing ground and soil temperature. These data will improve process model predictions of streamflow from the watershed.

  2. Characterising the structural properties of polymer separators for lithium-ion batteries in 3D using phase contrast X-ray microscopy

    NASA Astrophysics Data System (ADS)

    Finegan, Donal P.; Cooper, Samuel J.; Tjaden, Bernhard; Taiwo, Oluwadamilola O.; Gelb, Jeff; Hinds, Gareth; Brett, Dan J. L.; Shearing, Paul R.

    2016-11-01

    Separators are an integral component for optimising performance and safety of lithium-ion batteries; therefore, a clear understanding of how their microstructure affects cell performance and safety is crucial. Phase contrast X-ray microscopy is used here to capture the microstructures of commercial monolayer, tri-layer, and ceramic-coated lithium-ion battery polymer separators. Spatial variations in key structural parameters, including porosity, tortuosity factor and pore size distribution, are determined through the application of 3D quantification techniques and stereology. The architectures of individual layers in multi-layer membranes are characterised, revealing anisotropy in porosity, tortuosity factor and mean pore size of the three types of separator. Detailed structural properties of the individual layers of multi-layered membranes are then related with their expected effect on safety and rate capability of cells.

  3. How to connect time-lapse recorded trajectories of motile microorganisms with dynamical models in continuous time

    NASA Astrophysics Data System (ADS)

    Pedersen, Jonas N.; Li, Liang; Grǎdinaru, Cristian; Austin, Robert H.; Cox, Edward C.; Flyvbjerg, Henrik

    2016-12-01

    We provide a tool for data-driven modeling of motility, data being time-lapse recorded trajectories. Several mathematical properties of a model to be found can be gleaned from appropriate model-independent experimental statistics, if one understands how such statistics are distorted by the finite sampling frequency of time-lapse recording, by experimental errors on recorded positions, and by conditional averaging. We give exact analytical expressions for these effects in the simplest possible model for persistent random motion, the Ornstein-Uhlenbeck process. Then we describe those aspects of these effects that are valid for any reasonable model for persistent random motion. Our findings are illustrated with experimental data and Monte Carlo simulations.

  4. Time-lapse CO2 monitoring using ambient-noise seismic interferometry: a feasibility study from Ketzin, Germany

    NASA Astrophysics Data System (ADS)

    Boullenger, Boris; Verdel, Arie; Paap, Bob; Thorbecke, Jan; Draganov, Deyan

    2015-04-01

    Seismic interferometry applied to ambient-noise measurements allows retrieval of the Green's function between two seismic receivers, by cross-correlating their recordings, as if from a source at one of the receivers. We propose to use ambient-noise seismic interferometry (ANSI) to retrieve reflection data. The time-lapse differences between different vintages of the retrieved data may help characterize property changes within a geologic reservoir with varying CO2 saturation. We test the feasibility of this time-lapse passive seismic method with numerical experiments based on the CO2-storage site of Ketzin, Germany. Ambient-noise recordings from Ketzin exhibit significant passive body-wave energy (