Time-lapse Image Series Research Articles

Apparatus design of operando hydrogen microscope for visualization of time-dependent distribution of hydrogen

The operando hydrogen microscope is an original apparatus that has been developed to visualize the time-dependent permeation of hydrogen through a sample. This apparatus is developed based on an ultrahigh vacuum scanning electron microscope (UHV SEM). The system consists of a lens system to focus ions produced by electron-stimulated desorption (ESD), an ion detector optimized for ESD-signal detection, and a two-dimensional measurement program synchronized with the position information of the electron beam. The developed detectors and electrostatic lenses enable highly sensitive detection of the ions. In this paper, we show the details of the instrumentation of the operando hydrogen microscope. We have succeeded in recording hydrogen and deuterium flowing from the rear of metals to the surface as a series of time-lapse images, showing the time-dependent changes in the surface distribution of hydrogen and deuterium. Deuterium permeation through the metal sample was dynamically visualized at the surface by the two-dimensional mapping of the desorbed ions induced by scanning electron beam irradiation.

Machine-Learning-Aided Quantification of Area Coverage of Adherent Cells from Phase-Contrast Images.

The advances in machine learning (ML) software availability, efficiency, and friendliness, combined with the increase in the computation power of personal computers, are harnessed to rapidly and (relatively) effortlessly analyze time-lapse image series of adherent cell cultures, taken with phase-contrast microscopy (PCM). Since PCM is arguably the most widely used technique to visualize adherent cells in a label-free, noninvasive, and nondisruptive manner, the ability to easily extract quantitative information on the area covered by cells, should provide a valuable tool for investigation. We demonstrate two cases, in one we monitor the shrinking of cells in response to a toxicant, and in the second we measure the proliferation curve of mesenchymal stem cells (MSCs).

Investigating the Performances of Wide-Field Raman Microscopy with Stochastic Optical Reconstruction Post-Processing.

Super-resolution fluorescence microscopy based on localization algorithms has tremendously impacted the field of imaging by improving the spatial resolution of optical measurements with specific blinking fluorophores and concomitant reduction of acquisition time. In vibrational spectroscopy and imaging, various methods have been developed to surpass the diffraction limit including near-field scattering methods, such as in tip-enhanced Raman and infrared spectroscopies. Although these scanning-probe techniques can provide exquisite spatial resolution, they often require long acquisition times and tedious fabrication of nano-scale scanning probes. Herein, stochastic optical reconstruction microscopy (STORM) protocol is applied on Raman measurements acquired using a wide-field home-built microscopy setup. We explore how the fluctuations of the Raman signal acquired over a series of time-lapse images at specific spectral ranges can be exploited with STORM processing, possibly revealing details with improved spatial resolution, under lower irradiance and with faster acquisition speed that cannot be achieved in point scanning mode over the same field of view. Samples studied here include patterned silicon, polystyrene microspheres on a silicon wafer, and graphene on a silicon/silicon dioxide substrate. The outcome presents an effective way to collect Raman images at selected spectral ranges with spatial resolutions of ∼200 nm over a large field of view under 532 nm excitation together with an acquisition speed improved by two orders of magnitude and under a significantly reduced irradiance compared to confocal laser scanning acquisition.

Video-based calcification assay: A novel method for kinetic analysis of osteogenesis in live cultures

Traditional methods of quantifying osteoblast calcification in culture require the use of calcium sensitive dyes, such as Arsenazo III or Alizarin Red S, which have been successfully used for decades to assess osteogenesis. Because these dyes elicit a colorimetric change when reacted with a cell lysate and are cytotoxic to live cells, they forfeit the ability to trace calcification longitudinally over time. Here, we demonstrate that image analysis and quantification of calcification can be performed from a series of time-lapse images acquired from videos. This method capitalizes on the unique facet of the mineralized extracellular matrix to appear black when viewed with phase contrast optics. This appearance of calcified areas had been previously documented to be characteristic to the formation of bone nodules in vitro. Due to this distinguishable appearance, extracting the information corresponding to calcification through segmentation allowed us to threshold only the pixels that comprise the mineralized areas in the image. Ultimately, this method can be used to quantify calcification yield, rates and kinetics facilitating the analyses of bone-supportive properties of growth factors and morphogens as well as of adverse effects elicited by toxicants. It may also be used on images that were acquired manually.•The method is less error-prone than absorption-based assays since it takes longitudinal measurements from the same cultures•It is cost effective as it foregoes the use of calcium-sensitive dyes•It is automatable and amenable to high-throughput and thus allows the concurrent quantification of multiple parameters of differentiation

QuoVadoPro, an Autonomous Tool for Measuring Intracellular Dynamics using Temporal Variance.

Trafficking of intracellular cargo is essential to cellular function and can be defective in pathological states including cancer and neurodegeneration. Tools to quantify intracellular traffic are thus necessary for understanding this fundamental cellular process, studying disease mechanisms, and testing the effects of therapeutic pharmaceuticals. In this article we introduce an algorithm called QuoVadoPro that autonomously quantifies the movement of fluorescently tagged intracellular cargo. QuoVadoPro infers the extent of intracellular motility based on the variance of pixel illumination in a series of time-lapse images. The algorithm is an unconventional approach to the automatic measurement of intracellular traffic and is suitable for quantifying movements of intracellular cargo under diverse experimental paradigms. QuoVadoPro is particularly useful to measure intracellular cargo movement in non-neuronal cells, where cargo trafficking occurs as short movements in mixed directions. The algorithm can be applied to images with low temporal or spatial resolutions and to intracellular cargo with varying shapes or sizes, like mitochondria or endoplasmic reticulum: situations in which conventional methods such as kymography and particle tracking cannot be applied. In this article we present a stepwise protocol for using the QuoVadoPro software, illustrate its methodology with common examples, discuss critical parameters for reliable data analysis, and demonstrate its use with a previously published example. © 2020 Wiley Periodicals LLC. Basic Protocol: QuoVadoPro, an autonomous tool for measuring intracellular dynamics using temporal variance.

Instance-Level Microtubule Tracking.

We propose a new method of instance-level microtubule (MT) tracking in time-lapse image series using recurrent attention. Our novel deep learning algorithm segments individual MTs at each frame. Segmentation results from successive frames are used to assign correspondences among MTs. This ultimately generates a distinct path trajectory for each MT through the frames. Based on these trajectories, we estimate MT velocities. To validate our proposed technique, we conduct experiments using real and simulated data. We use statistics derived from real time-lapse series of MT gliding assays to simulate realistic MT time-lapse image series in our simulated data. This data set is employed as pre-training and hyperparameter optimization for our network before training on the real data. Our experimental results show that the proposed supervised learning algorithm improves the precision for MT instance velocity estimation drastically to 71.3% from the baseline result (29.3%). We also demonstrate how the inclusion of temporal information into our deep network can reduce the false negative rates from 67.8% (baseline) down to 28.7% (proposed). Our findings in this work are expected to help biologists characterize the spatial arrangement of MTs, specifically the effects of MT-MT interactions.

Slow Relaxation of Shape and Orientational Texture in Membrane Gel Domains.

Gel domains in lipid bilayers are structurally more complex than fluid domains. Growth dynamics can lead to noncircular domains with a heterogeneous orientational texture. Most model membrane studies involving gel domain morphology and lateral organization assume the domains to be static. Here we show that rosette shaped gel domains, with heterogeneous orientational texture and a central topological defect, after early stage growth, undergo slow relaxation. On a time scale of days to weeks domains converge to circular shapes and approach uniform texture. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) enriched gel domains are grown by cooling 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DPPC bilayers into the solid-liquid phase coexistence region and are visualized with fluorescence microscopy. The relaxation of individual domains is quantified through image analysis of time-lapse image series. We find a shape relaxation mechanism which is inconsistent with Ostwald ripening and coalescence as observed in membrane systems with coexisting liquid phases. Moreover, domain texture changes in parallel with the changes in domain shape, and selective melting and growth of particular subdomains cause the texture to become more uniform. We propose a relaxation mechanism based on relocation of lipids from high-energy lattice positions, through evaporation-condensation and edge diffusion, to low-energy positions. The relaxation process is modified significantly by binding Shiga toxin, a bacterial toxin from Shigella dysenteriae, to the membrane surface. Binding alters the equilibrium shape of the gel domains from circular to eroded rosettes with disjointed subdomains. This observation may be explained by edge diffusion while evaporation-condensation is restricted, and it provides further support for the proposed overall relaxation mechanism.

Quantitative imaging of focal adhesion dynamics and their regulation by HGF and Rap1 signaling

Cell migration is crucial in development, tissue repair and immunity and frequently aberrant in pathological processes including tumor metastasis. Focal adhesions (FAs) are integrin-based adhesion complexes that form the link between the cytoskeleton and the extracellular matrix and are thought to orchestrate cell migration. Understanding the regulation of FAs by (oncogenic) signaling pathways may identify strategies to target pathological cell migration. Here we describe the development of a robust FA tracker that enables the automatic, multi-parametric analysis of FA dynamics, morphology and composition from time-lapse image series generated by total internal reflection fluorescence (TIRF) microscopy. In control prostate carcinoma cells, this software recapitulates previous findings that relate morphological characteristics of FAs to their lifetime and their cellular location. We then investigated how FAs are altered when cell migration is induced by the metastasis-promoting hormone HGF and subsequently inhibited by activation of the small GTPase Rap1. We performed a detailed analysis of individual FA parameters, which identified FA size, sliding and intensity as primary targets of Rap1. HGF did not have strong effects on any of the FA parameters within the first hours of its addition. Subsequent Bayesian network inference (BNI), using all measured parameters as input, revealed little correlation between changes in cell migration and FA characteristics in this prostate carcinoma cell line. Instead BNI indicated a concerted coordination of cell size and FA parameters. Thus our results did not reveal a direct relation between the regulation of cell migration and the regulation of FA dynamics.

Liquid scanning transmission electron microscopy: Nanoscale imaging in micrometers-thick liquids

Abstract Scanning transmission electron microscopy (STEM) of specimens in liquid is possible using a microfluidic chamber with thin silicon nitride windows. This paper includes an analytic equation of the resolution as a function of the sample thickness and the vertical position of an object in the liquid. The equipment for STEM of liquid specimen is briefly described. STEM provides nanometer resolution in micrometer-thick liquid layers with relevance for both biological research and materials science. Using this technique, we investigated tagged proteins in whole eukaryotic cells, and gold nanoparticles in liquid with time-lapse image series. Possibly future applications are discussed.

Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells

Molecular mechanisms underlying the effects of Fyn on cell morphology, pseudopodium movement, and cell migration were investigated. The Fyn gene was subcloned into pEGFP-N1 to produce pEGFP-N1-Fyn. Chinese hamster ovary (CHO) cells were transfected with pEGFP-N1-Fyn. The expression of Fyn mRNA and proteins was monitored by reverse transcription-PCR and Western blotting. Additionally, transfected cells were stained with 4',6-diamidino-2-phenylindole and a series of time-lapse images was taken. Sequences of the recombinant plasmids pMD18-T-Fyn and pEGFP-N1-Fyn were confirmed by sequence identification using National Center for Biotechnology Information in USA, and Fyn expression was detected by RT-PCR and Western blotting. The morphology of CHO cells transfected with the recombinant vector was significantly altered. Fyn expression induced filopodia and lamellipodia formation. Based on these results, we concluded that overexpression of mouse Fyn induces the formation of filopodia and lamellipodia in CHO cells, and promotes cell movement.

Quantitative Analysis of Colony Morphology in Yeast

Microorganisms often form multicellular structures such as biofilms and structured colonies that can influence the organism's virulence, drug resistance, and adherence to medical devices. Phenotypic classification of these structures has traditionally relied on qualitative scoring systems that limit detailed phenotypic comparisons between strains. Automated imaging and quantitative analysis have the potential to improve the speed and accuracy of experiments designed to study the genetic and molecular networks underlying different morphological traits. For this reason, we have developed a platform that uses automated image analysis and pattern recognition to quantify phenotypic signatures of yeast colonies. Our strategy enables quantitative analysis of individual colonies, measured at a single time point or over a series of time-lapse images, as well as the classification of distinct colony shapes based on image-derived features. Phenotypic changes in colony morphology can be expressed as changes in feature space trajectories over time, thereby enabling the visualization and quantitative analysis of morphological development. To facilitate data exploration, results are plotted dynamically through an interactive Yeast Image Analysis web application (YIMAA; http://yimaa.cs.tut.fi) that integrates the raw and processed images across all time points, allowing exploration of the image-based features and principal components associated with morphological development.

Computer Aided Alignment and Quantitative 4D Structural Plasticity Analysis of Neurons

The rapid development of microscopic imaging techniques has greatly facilitated time-lapse imaging of neuronal morphology. However, analysis of structural dynamics in the vast amount of 4-Dimensional data generated by in vivo or ex vivo time-lapse imaging still relies heavily on manual comparison, which is not only laborious, but also introduces errors and discrepancies between individual researchers and greatly limits the research pace. Here we present a supervised 4D Structural Plasticity Analysis (4D SPA) computer method to align and match 3-Dimensional neuronal structures across different time points on a semi-automated basis. We demonstrate 2 applications of the method to analyze time-lapse data showing gross morphological changes in dendritic arbor morphology and to identify the distribution and types of branch dynamics seen in a series of time-lapse images. Analysis of the dynamic changes of neuronal structure can be done much faster and with greatly improved consistency and reliability with the 4D SPA supervised computer program. Users can format the neuronal reconstruction data to be used for this analysis. We provide file converters for Neurolucida and Imaris users. The program and user manual are publically accessible and operate through a graphical user interface on Windows and Mac OSX.

Automated image analysis for tracking cargo transport in axons

The dynamics of cargo movement in axons encodes crucial information about the underlying regulatory mechanisms of the axonal transport process in neurons, a central problem in understanding many neurodegenerative diseases. Quantitative analysis of cargo dynamics in axons usually includes three steps: (1) acquiring time-lapse image series, (2) localizing individual cargos at each time step, and (3) constructing dynamic trajectories for kinetic analysis. Currently, the later two steps are usually carried out with substantial human intervention. This article presents a method of automatic image analysis aiming for constructing cargo trajectories with higher data processing throughput, better spatial resolution, and minimal human intervention. The method is based on novel applications of several algorithms including 2D kymograph construction, seed points detection, trajectory curve tracing, back-projection to extract spatial information, and position refining using a 2D Gaussian fitting. This method is sufficiently robust for usage on images with low signal-to-noise ratio, such as those from single molecule experiments. The method was experimentally validated by tracking the axonal transport of quantum dot and DiI fluorophore-labeled vesicles in dorsal root ganglia neurons.

Meiotic chromosome pairing and bouquet formation during Eimeria tenella sporulation

In Eimeria tenella, meiotic division occurs exclusively in oocysts within the first 8 h of sporulation. Difficulties with the wall-oocyst breakage in gaining access to chromosomes during meiosis have resulted in a scarcity of morphological data on Eimeria chromosomes. This study tracks the general behaviour of telomeres, attachment plaques and synaptonemal complexes in the nucleus of the meiotic oocyst of E. tenella. Fluorescence microscopy methods, in combination with immunoelectron microscopy techniques, were applied to obtain a series of time-lapse images during oocyst sporulation. Antibodies to Structural Maintenance of Chromosome proteins SMC1 and SMC3, and lamin were labelled with either fluorescence or colloidal gold to visualise the telomeres, central elements of the synaptonemal complex (SC) and nuclear periphery, respectively, at both the structural and ultrastructural levels. Using oocyst spreads and ultrathin sections of fixed oocysts it was possible to study telomere dynamics at stages during meiosis. The stages of the meiotic prophase I are delineated on the basis of the telomere position and the SC synapsis and desynapsis. During the leptotene stage, at 4 h following the start of sporulation, meiotic chromosomes attached to the nuclear envelope. At that stage, chromosome synapsis was initiated in the telomeric regions but no interstitial synapsis pairing was observed. In the zygotene stage, telomere signals were clustered in a limited area of the nuclear envelope. Bouquet formation occurred at 5 h after the start of sporulation, whereas chromosomes did not appear completely synapsed until the pachytene stage at 6 h of sporulation. Desynapsis was observed at 8 h of sporulation during the diplotene stage. This study provides the first morphological description of both the behaviour of the chromosomes and the timing of the prophase I stages in the meiotic nucleus of E. tenella.

Videomicroscopic extraction of specific information on cell proliferation and migration in vitro

In vitro cell imaging is a useful exploratory tool for cell behavior monitoring with a wide range of applications in cell biology and pharmacology. Combined with appropriate image analysis techniques, this approach has been shown to provide useful information on the detection and dynamic analysis of cell events. In this context, numerous efforts have been focused on cell migration analysis. In contrast, the cell division process has been the subject of fewer investigations. The present work focuses on this latter aspect and shows that, in complement to cell migration data, interesting information related to cell division can be extracted from phase-contrast time-lapse image series, in particular cell division duration, which is not provided by standard cell assays using endpoint analyses. We illustrate our approach by analyzing the effects induced by two sigma-1 receptor ligands (haloperidol and 4-IBP) on the behavior of two glioma cell lines using two in vitro cell models, i.e., the low-density individual cell model and the high-density scratch wound model. This illustration also shows that the data provided by our approach are suggestive as to the mechanism of action of compounds, and are thus capable of informing the appropriate selection of further time-consuming and more expensive biological evaluations required to elucidate a mechanism.

Bacterial Actin Assembly Requires Toca-1 to Relieve N-WASP Autoinhibition

Actin polymerization in the mammalian cytosol can be locally activated by mechanisms that relieve the autoinhibited state of N-WASP, an initiator of actin assembly, a process that also requires the protein Toca-1. Several pathogenic bacteria, including Shigella, exploit this host feature to infect and disseminate efficiently. The Shigella outer membrane protein IcsA recruits N-WASP, which upon activation at the bacterial surface mediates localized actin polymerization. The molecular role of Toca-1 in N-WASP activation during physiological or pathological actin assembly processes in intact mammalian cells remains unclear. We show that actin tail initiation by S. flexneri requires Toca-1 for the conversion of N-WASP from a closed inactive conformation to an open active one. While N-WASP recruitment is dependent on IcsA, Toca-1 recruitment is instead mediated by S. flexneri type III secretion effectors. Thus, S. flexneri independently hijacks two nodes of the N-WASP actin assembly pathway to initiate localized actin tail assembly.

The effect of flow direction and magnitude on the initial distribution of Pseudomonas aeruginosa cells attached to glass

The effect of flowrate and Reynolds Number, Re, on the spatial distribution of individual Pseudomonas aeruginosa cells during their initial attachment to glass flowcells was observed in a series of time-lapse images obtained over a 56-h period. It was shown that flow affected the distribution at Re > 245. Under laminar flow conditions, Re = 96, the distribution of bacterial cells in 200 sub-areas was accurately predicted by using the Poisson distribution and was not dependent on the orientation or shape of the sub-areas. Under turbulent flow conditions, Re = 2220, cells initially attached in streaks along the line of flow. As bacterial cells accumulated on the surface, the streaks broadened and the distribution became more uniform. Analyses showed that, initially, flow had an effect on cell distribution in the flowcell with Re = 245, with significantly greater effects at higher Re. As the cell surface densities increased, the effect of flow direction on cell distribution decreased. It is concluded that the visco-elastic properties of the extracellular polymeric substances (EPS) in which the cells are embedded, significantly affect the distribution of attaching cells.

Dynamics of β1-Integrins in Living Fibroblasts—Effect of Substratum Wettability

The dynamics of integrin receptors mobility was studied in living human fibroblasts using fluorescence-labeled β1-integrin monoclonal antibodies. Time-lapse image series were obtained by confocal laser scanning microscopy when cells were adhering on model hydrophilic (clean glass) and hydrophobic (octadecyl-silanized; i.e., ODS) surfaces coated with fibronectin. Direct measurements showed approximately twice-higher velocity of integrins on glass compared to ODS, and these velocities varied in different zones of the cells. A kinetic model and algorithm for quantification of images was developed, and the analysis identified three receptor populations on glass: immobilized (82.76% of all), slow (4.16%), and fast (13.08%), while, on ODS, only two were identified: immobilized (83.36%) and fast (16.64%). Fast integrins in the peripheral zone of cells have maximal velocities of 0.353±0.02μm/min (n=48, four cells) on hydrophilic and 0.218±0.02μm/min (n=30, three cells) on hydrophobic substrata. The slow population has a velocity of 0.114μm/min (n=48, four cells). Further analyses show that these velocities also differ significantly in the peripheral and middle zones of cells in a substrate-dependent fashion. A well-defined circular motion of receptors around the cell center expressed mainly on hydrophobic substrata was monitored and quantified as well.

Recent Highlights from Atomic Force Microscopy of DNA

Seven recent highlights are presented from atomic force microscopy (AFM) of DNA in this lab. The first two involve advances in the observation of enzymatic reactions in near-physiological solutions. E. coli RNA polymerase was observed to process along its DNA template in a series of time-lapse images [S. Kasas, et al., Biochemistry 36, 461 (1997)], and a new small-cantilever atomic force microscope (AFM) imaged DNA degradation by DNase I at rates as fast as two seconds per image. The next five highlights involve structural observations of DNA and DNA-protein complexes, including DNA condensed for gene delivery, sequence-dependent DNA condensation, an AFM assay for RNA polymerase, and AFM evidence for a yeast kinetochore complex that may be involved in holding together sister chromatids during cell division.

A Non-Invasive Solidification Sensor for Metal Casting

A non-invasive sensor based on high-energy x-ray computed tomography has been developed for monitoring the progression of solidification in metal casting. The sensor comprised a 6 MeV linear accelerator and a 128-channel CdWO 4 detector array. Laboratory experiments were performed on cylindrical castings of pure aluminum and its alloys. The sensor produced a series of time-lapse images showing the movement of the liquid/solid interface during solidification. The solidification front was imaged every two minutes with a planar spatial resolution of 1.6 mm and a contrast sensitivity of 1.5%. The tomography results agreed well with those obtained by thermocouple measurements.