Analysis of surface growth in shoot apices

A salient feature of shoot meristem growth is the maintenance of distinct anatomical and morphological features despite a continuous flux of cells. To investigate how meristem organization is self-perpetuated, we developed a protocol for the analysis of meristem growth in 3-D. Our protocol uses a non-destructive replica method to follow the pattern of cell expansion and cell divisions on the meristem surface over several days. Algorithms to reconstruct the meristem surface and compute its curvature and rate of extension were implemented. We applied this approach to the shoot apical meristem of Anagallis arvensis and showed that a subcellular resolution of extension rates can be achieved. This is the first detailed quantitative analysis of meristem geometry and surface expansion in 3-D. This new approach will be useful to connect cellular activities such as cell expansion, cell division, and differential gene expression with overall meristem morphogenesis.     

cytoNet: Spatiotemporal network analysis of cell communities

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet’s capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.     

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology¹ even in complex tissue sections². Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells³, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.     

Three-dimensional reconstruction of Picea wilsonii Mast. pollen grains using automated electron microscopy

Three-dimensional electron microscopy(3 D-EM) has attracted considerable attention because of its ability to provide detailed information with respect to developmental analysis. However, large-scale high-resolution 3 D reconstruction of biological samples remains challenging. Herein, we present a 3 D view of a Picea wilsonii Mast. pollen grain with 100 nm axial and38.57 nm lateral resolution using AutoCUTS-SEM(automatic collector of ultrathin sections-scanning electron microscopy). We established a library of 3,127 100 nm thick serial sections of pollen grains for preservation and observation, demonstrating that the protocol can be used to analyze large-volume samples. After obtaining the SEM images, we reconstructed an entire pollen grain comprising 734 serial sections. The images produced by 3D reconstruction clearly revealed the main components of the P.wilsonii pollen grain, i.e., two sacci and pollen corpus, tube cell, generative cell, and two degenerated prothallial cells, and their internal organization. In addition, we performed a quantitative analysis of the different pollen grain cells, including sacci, and found that there were 202 connections within a saccus SEM image. Thus, for the first time, this study provided a global 3D view of the entire pollen grain, which will be useful for analyzing pollen development and growth.     

Integrating intracellular dynamics using CompuCell3D and Bionetsolver: applications to multiscale modelling of cancer cell growth and invasion

In this paper we present a multiscale, individual-based simulation environment that integrates CompuCell3D for lattice-based modelling on the cellular level and Bionetsolver for intracellular modelling. CompuCell3D or CC3D provides an implementation of the lattice-based Cellular Potts Model or CPM (also known as the Glazier-Graner-Hogeweg or GGH model) and a Monte Carlo method based on the metropolis algorithm for system evolution. The integration of CC3D for cellular systems with Bionetsolver for subcellular systems enables us to develop a multiscale mathematical model and to study the evolution of cell behaviour due to the dynamics inside of the cells, capturing aspects of cell behaviour and interaction that is not possible using continuum approaches. We then apply this multiscale modelling technique to a model of cancer growth and invasion, based on a previously published model of Ramis-Conde et al. (2008) where individual cell behaviour is driven by a molecular network describing the dynamics of E-cadherin and β-catenin. In this model, which we refer to as the centre-based model, an alternative individual-based modelling technique was used, namely, a lattice-free approach. In many respects, the GGH or CPM methodology and the approach of the centre-based model have the same overall goal, that is to mimic behaviours and interactions of biological cells. Although the mathematical foundations and computational implementations of the two approaches are very different, the results of the presented simulations are compatible with each other, suggesting that by using individual-based approaches we can formulate a natural way of describing complex multi-cell, multiscale models. The ability to easily reproduce results of one modelling approach using an alternative approach is also essential from a model cross-validation standpoint and also helps to identify any modelling artefacts specific to a given computational approach.     

Rayburst sampling, an algorithm for automated three-dimensional shape analysis from laser scanning microscopy images

Precise quantification of complex three-dimensional (3D) structures from laser scanning microscopy (LSM) images is increasingly necessary in understanding normal function and pathologic processes in biology. This protocol describes a versatile shape analysis algorithm, Rayburst sampling, that generates automated 3D measurements from LSM images. Rayburst defines and efficiently casts a multidirectional core of rays from an interior point to the surface of a solid, allowing precise quantification of anisotropic and irregularly shaped 3D structures. Quantization error owing to the finite voxel representation in digital images is minimized by interpolating intensity values continuously between voxels. The Rayburst algorithm provides a primitive for the development of higher level algorithms that solve specific shape analysis problems. Examples are provided of applications to 3D neuronal morphometry: (i) estimation of diameters in tubular neuronal dendritic branching structures, and (ii) measurement of volumes and surface areas for dendritic spines and spatially complex histopathologic structures.     

The effect of MMP inhibitor GM6001 on early fibroblast-mediated collagen matrix contraction is correlated to a decrease in cell protrusive activity

Although fibroblasts play an essential part during the wound healing response, the mechanisms by which they mediate tissue remodelling and contraction are still unclear. Using live cell and matrix imaging within 3D free-floating fibroblast-populated collagen lattices as a model for tissue contraction, we compared the behaviour of a range of fibroblasts with low and high contraction abilities and analysed the effect of the broad spectrum MMP-inhibitor GM6001 on cell behaviour and matrix contraction. We identified two mechanisms underlying matrix contraction, one via direct cell-mediated contractile activity, the second through matrix degradation. These appear to be linked to cell morphology and regulated by the collagen concentration within the matrix. Cells with a rounded morphology proliferated in the matrix but did not remodel it efficiently, resulting in a poor ability to contract matrices. Cells with an elongated morphology showed higher levels of protrusive activity, leading to efficient matrix remodelling and contraction. GM6001 inhibited week-long matrix contraction to various extents with the different cell lines. However, quantitative analysis of the cell protrusive activity showed that GM6001 consistently decreased cell dynamics in 3D by about 20%, and this was correlated with a significant reduction in early matrix contraction. Overall our results suggest that although fibroblast-mediated matrix contraction depends on both cell dynamics and MMP-mediated matrix degradation, the efficiency of GM6001 treatment in preventing contraction might be linked to a direct effect on cell dynamics.     

Technical advance: near-infrared femtosecond laser pulses as a novel non-invasive means for dye-permeation and 3D imaging of localised dye-coupling in the Arabidopsis root meristem

We have used near-infrared femtosecond Titanium: Sapphire laser pulses as novel non-invasive means for dye loading into various cell types of the Arabidopsis root meristem, and by 3D imaging have assessed the extent of dye coupling between the meristematic cells. The post-embryonic primary root of Arabidopsis thaliana has an invariant ontogeny and fixed cellular organisation which makes it an attractive model system to study developmental events involving cell fate determination, cellular differentiation and pattern formation. Local intercellular communication and local transmission of positional signals are likely to play a pivotal role in cell proliferation and regulation of differentiation. We have therefore examined the extent to which the constituent cells in the root meristem are symplastically coupled. Following laser-assisted loading of membrane impermeate fluorescent dye propidium iodide (PI) in single cells, we show by time-lapse and 3D imaging that in the root tip all undifferentiated cells are dye-coupled. When PI is permeated into the central cells, it rapidly moved into the adjacent initials of the columella, cortex, pericycle and stele. Interestingly, when only either of the initials were loaded with the dye, it never moved into any of the central cells. Amongst the epidermal cells, the differentiated hair cells are symplastically isolated. Our data provide evidence (1) for differential dye-coupling behaviour between quiescent centre cells and the neighbouring initials; (2) that cells in the root are coupled during stages at which the cell-lineage pattern is formed and that it becomes progressively secluded as they differentiate and the pattern is fixed. Taken together, our NIR-laser mediated approach is highly efficient and has numerous potential applications for non-invasive permeation of dyes in different cell types.     

Dynamic and compensatory responses of Arabidopsis shoot and floral meristems to CLV3 signaling

In Arabidopsis thaliana, the stem cell population of the shoot system is controlled by regulatory circuitry involving the WUSCHEL (WUS) and CLAVATA (CLV1-3) genes. WUS signals from the organizing center (OC) to promote stem cell fate at the meristem apex. Stem cells express the secreted peptide CLV3 that activates a signal transduction cascade to restrict WUS expression, thus providing a feedback mechanism. Stem cell homeostasis is proposed to be achieved by balancing these signals. We tested the dynamics of CLV3 signaling using an inducible gene expression system. We show here that increasing the CLV3 signal can very rapidly repress WUS expression during development, which in turn causes a fast reduction of CLV3 expression. We demonstrate that increased CLV3 signaling restricts meristem growth and promotes allocation of peripheral meristem cells into organ primordia. In addition, we extend the current model for stem cell control by showing that meristem homeostasis tolerates variation in CLV3 levels over a 10-fold range and that high-level CLV3 signaling can be partially compensated with time, indicating that the level of CLV3 expression communicates only limited information on stem cell number to the underlying OC cells.     

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.     

Resolving cargo-motor-track interactions with bifocal parallax single-particle tracking

Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single-particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0–360°), polar angle, and three-dimensional (3D) displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only were well-known active intracellular transport and free diffusion observed, but new interactions (tight attachment and tethered rotation) were also discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.     

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.     

The iRoCS Toolbox – 3D analysis of the plant root apical meristem at cellular resolution

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three‐dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single‐cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open‐source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem.     

Three-Dimensional Quantification of Spheroid Degradation-Dependent Invasion and Invadopodia Formation

Invadopodia are actin-rich, proteolytic structures that enable cancer cell to invade into the surrounding tissues. Several in vitro invasion assays have been used in the literature ranging from directional quantitative assays to complex three-dimensional (3D) analyses. One of the main limitations of these assays is the lack of quantifiable degradation-dependent invasion in a three-dimensional (3D) environment that mimics the tumor microenvironment. In this article, we describe a new invasion and degradation assay based on the currently available tumor spheroid model that allows long-term high-resolution imaging of the tumor, precise quantification, and visualization of matrix degradation and multichannel immunocytochemistry. By incorporating a degradation marker (DQ-Green BSA) into a basement-membrane matrix, we demonstrate the ability to quantitate cancer cell-induced matrix degradation in 3D. Also, we describe a technique to generate histological sections of the tumor spheroid allowing the detection of invadopodia formation in the 3D tumor spheroid. This new technique provides a clear advantage for studying cancer in vitro and will help address critical questions regarding the dynamics of cancer cell invasion.     

Label-free observation of three-dimensional morphology change of a single PC12 cell by digital holographic microscopy

Observation of three-dimensional (3D) morphology changes of a single mammalian cell is very useful to understand cell response for various stimuli. Conventional techniques to evaluate morphology changes with sufficient precision and high temporal resolution are limited. For example, the confocal fluorescence microscope is available to take 3D morphology changes, whereas fluorescence microscopic observation requires labeling the cells with fluorescence dye. Recently, a novel imaging method based on digital holography was developed for nonlabeling microscopic observation of 3D morphology. Digital holographic microscopy has high potentiality in digital focusing properties, video-frequency capability, noninvasive operation, and so forth. It obtains a quantitative phase image of a living cell from a single recorded hologram, with interferometric accuracy, and surveys the rapid morphology change of a single cell. In this study, digital holographic microscopy was applied to monitor the 3D morphology change of an individual PC12 cell, a nerve model cell, subjected to high K(+) stimulation. Phase images of the rapidly swelling cell were acquired, and time lapse reconstruction of 3D cell morphology was performed from phase images. Our results demonstrate that digital holographic imaging is a powerful new tool for evaluation of cell response against various stimulants without any labeling reagent.     

Banana (Musa spp.) as a model to study the meristem proteome: acclimation to osmotic stress

Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop.     

A three-dimensional insight into the complexity of flow convergence in mitral regurgitation: adjunctive benefit of anatomic regurgitant orifice area

Mitral effective regurgitant orifice area (EROA) using the flow convergence (FC) method is used to quantify the severity of mitral regurgitation (MR). However, it is challenging and prone to interobserver variability in complex valvular pathology. We hypothesized that real-time three-dimensional (3D) transesophageal echocardiography (RT3D TEE) derived anatomic regurgitant orifice area (AROA) can be a reasonable adjunct, irrespective of valvular geometry. Our goals were to 1) to determine the regurgitant orifice morphology and distance suitable for FC measurement using 3D computational flow dynamics and finite element analysis (FEA), and (2) to measure AROA from RT3D TEE and compare it with 2D FC derived EROA measurements. We studied 61 patients. EROA was calculated from 2D TEE images using the 2D-FC technique, and AROA was obtained from zoomed RT3DE TEE acquisitions using prototype software. 3D computational fluid dynamics by FEA were applied to 3D TEE images to determine the effects of mitral valve (MV) orifice geometry on FC pattern. 3D FEA analysis revealed that a central regurgitant orifice is suitable for FC measurements at an optimal distance from the orifice but complex MV orifice resulting in eccentric jets yielded nonaxisymmetric isovelocity contours close to the orifice where the assumptions underlying FC are problematic. EROA and AROA measurements correlated well (r = 0.81) with a nonsignificant bias. However, in patients with eccentric MR, the bias was larger than in central MR. Intermeasurement variability was higher for the 2D FC technique than for RT3DE-based measurements. With its superior reproducibility, 3D analysis of the AROA is a useful alternative to quantify MR when 2D FC measurements are challenging.     

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM)

The development of three‐dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label‐free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion‐relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass‐transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.     

Intracellular Localization of 3H-IAA in the Apical Bud of Lycopersicon esculentum

Driss-Ecole, D. and Perbal, G. 1987. Intracellular localizationof ³H-IAA in the apical bud of Lycopersicon esculentum.—J.exp. Bot. 38: 1362–1372. High resolution autoradiography of ³H-IAA was performed on ultra-thinsections of the apical bud of Lycopersicon esculentum treatedby DCC or glutaraldehyde. A quantitative study of the localizationof the labelling in the compartments (cell wall, cytoplasm,vacuoles and nucleus) of four types of cells (cells of the lateralzone of the apex, cells of the pith meristem, proximal and distalcells of the upper part of the pith) was made. The statisticalanalysis of the results has proved that the variability of thelabelling of the cell compartments in the four cell types wassimilar after treatment by DCC or glutaraldehyde. The densityof labelling is higher in each compartment of the meristematiccells compared with those of the differentiating cells. Thecells of the pith meristem can be distinguished from the meristematiccells of the lateral zone of the apex by a greater density oflabelling in the cytoplasm and the nucleus. These two cellulartypes contain a high density of radioactivity in vacuoles. Byconsidering the percentage of labelling of each compartmentrelative to the total labelling in the cell, it can be shownthat the meristematic cells are characterized by a high percentagein the nucleus whereas the vacuoles of the differentiating cellscontain the highest percentage of radioactivity. Key words: ³H-IAA, tomato shoot