scholarly journals Cell Tracking Profiler – a user-driven analysis framework for evaluating 4D live-cell imaging data

2020 ◽  
Vol 133 (22) ◽  
pp. jcs241422
Author(s):  
Claire Mitchell ◽  
Lauryanne Caroff ◽  
Jose Alonso Solis-Lemus ◽  
Constantino Carlos Reyes-Aldasoro ◽  
Alessandra Vigilante ◽  
...  
Keyword(s):  
Image Analysis ◽  
Cell Tracking ◽  
Cell Function ◽  
Ground Truth ◽  
Time Lapse ◽  
Automated Image Analysis ◽  
Imaging Data ◽  
Cell Behaviour ◽  
Muscle Stem Cell

ABSTRACTAccurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo. Here, by using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm, we established a ‘ground truth’ for muSC behaviour. This revealed that segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user-defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) is a package that runs two existing programs, HK Means and Phagosight within the Icy image analysis suite, to enable user-managed cell tracking from 3D time-lapse datasets to provide measures of cell shape and movement. We demonstrate how CTP can be used to reveal changes to cell behaviour of muSCs in response to manipulation of the cell cytoskeleton by small-molecule inhibitors. CTP and the associated tools we have developed for analysis of outputs thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets that are not amenable to straightforward analysis.

scholarly journals Cell Tracking Profiler: a user-driven analysis framework for evaluating 4D live cell imaging data

2019 ◽  
Author(s):  
Claire Mitchell ◽  
Lauryanne Caroff ◽  
Alessandra Vigilante ◽  
Jose Alonso Solis-Lemus ◽  
Constantino Carlos Reyes-Aldasoro ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Shape ◽  
Cell Tracking ◽  
Cell Function ◽  
Ground Truth ◽  
Automated Image Analysis ◽  
Imaging Data ◽  
Cell Behaviour ◽  
Reduction Methods

AbstractAccurate measurements of cell morphology and behaviour are fundamentally important for understanding how disease, molecules and drugs affect cell function in vivo. Using muscle stem cell (muSC) responses to injury in zebrafish as our biological paradigm we have established a ground truth for muSC cell behaviour. This revealed that variability in segmentation and tracking algorithms from commonly used programs are error-prone, leading us to develop a fast semi-automated image analysis pipeline that allows user defined parameters for segmentation and correction of cell tracking. Cell Tracking Profiler (CTP) operates through the freely available Icy platform, and allows user-managed cell tracking from 3D time-lapsed datasets to provide measures of cell shape and movement. Using dimensionality reduction methods, multiple correlation and regression analyses we identify myosin II-dependent parameters of muSC behaviour during regeneration. CTP and the associated statistical tools we have developed thus provide a powerful framework for analysing complex cell behaviour in vivo from 4D datasets.SummaryAnalysis of cell shape and movement from 3D time-lapsed datasets is currently very challenging. We therefore designed Cell Tracking Profiler for analysing cell behaviour from complex datasets and demonstrate its effectiveness by analysing stem cell behaviour during muscle regeneration in zebrafish.

scholarly journals Tracking calcium dynamics from individual neurons in behaving animals

2021 ◽  
Vol 17 (10) ◽  
pp. e1009432
Author(s):  
Thibault Lagache ◽  
Alison Hanson ◽  
Jesús E. Pérez-Ortega ◽  
Adrienne Fairhall ◽  
Rafael Yuste
Keyword(s):  
Visual Cortex ◽  
Calcium Imaging ◽  
Functional Characterization ◽  
Ground Truth ◽  
Time Lapse ◽  
Local Deformation ◽  
Neuron Activity ◽  
Imaging Data ◽  
Wide Range

Measuring the activity of neuronal populations with calcium imaging can capture emergent functional properties of neuronal circuits with single cell resolution. However, the motion of freely behaving animals, together with the intermittent detectability of calcium sensors, can hinder automatic monitoring of neuronal activity and their subsequent functional characterization. We report the development and open-source implementation of a multi-step cellular tracking algorithm (Elastic Motion Correction and Concatenation or EMC2) that compensates for the intermittent disappearance of moving neurons by integrating local deformation information from detectable neurons. We demonstrate the accuracy and versatility of our algorithm using calcium imaging data from two-photon volumetric microscopy in visual cortex of awake mice, and from confocal microscopy in behaving Hydra, which experiences major body deformation during its contractions. We quantify the performance of our algorithm using ground truth manual tracking of neurons, along with synthetic time-lapse sequences, covering a wide range of particle motions and detectability parameters. As a demonstration of the utility of the algorithm, we monitor for several days calcium activity of the same neurons in layer 2/3 of mouse visual cortex in vivo, finding significant turnover within the active neurons across days, with only few neurons that remained active across days. Also, combining automatic tracking of single neuron activity with statistical clustering, we characterize and map neuronal ensembles in behaving Hydra, finding three major non-overlapping ensembles of neurons (CB, RP1 and RP2) whose activity correlates with contractions and elongations. Our results show that the EMC2 algorithm can be used as a robust and versatile platform for neuronal tracking in behaving animals.

scholarly journals Machine learning-based classification of mitochondrial morphology in primary neurons and brain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Garrett M. Fogo ◽  
Anthony R. Anzell ◽  
Kathleen J. Maheras ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Image Analysis ◽  
Cortical Neurons ◽  
Mitochondrial Dynamics ◽  
Automated Image Analysis ◽  
Mitochondrial Morphology ◽  
Analysis Pipeline ◽  
Genetic Ablation ◽  
Block Face

AbstractThe mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

scholarly journals Engineered Heterochronic Parabiosis in 3D Microphysiological System for Identification of Muscle Rejuvenating Factors

2020 ◽  
Author(s):  
Yunki Lee ◽  
Jeongmoon J. Choi ◽  
Song Ih Ahn ◽  
Nan Hee Leea ◽  
Woojin M. Han ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Three Dimensional ◽  
Muscle Stem Cell ◽  
Organ Systems ◽  
Systemic Factors ◽  
Underlying Mechanisms ◽  
On Chip

AbstractExposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

scholarly journals Inference of Synaptic Connectivity and External Variability in Neural Microcircuits

2019 ◽  
Author(s):  
Cody Baker ◽  
Emmanouil Froudarakis ◽  
Dimitri Yatsenko ◽  
Andreas S. Tolias ◽  
Robert Rosenbaum
Keyword(s):  
Functional Connectivity ◽  
Large Scale ◽  
Network Models ◽  
Ground Truth ◽  
External Input ◽  
Synaptic Connectivity ◽  
Imaging Data ◽  
Common Input ◽  
Simplifying Assumptions

AbstractA major goal in neuroscience is to estimate neural connectivity from large scale extracellular recordings of neural activity in vivo. This is challenging in part because any such activity is modulated by the unmeasured external synaptic input to the network, known as the common input problem. Many different measures of functional connectivity have been proposed in the literature, but their direct relationship to synaptic connectivity is often assumed or ignored. For in vivo data, measurements of this relationship would require a knowledge of ground truth connectivity, which is nearly always unavailable. Instead, many studies use in silico simulations as benchmarks for investigation, but such approaches necessarily rely upon a variety of simplifying assumptions about the simulated network and can depend on numerous simulation parameters. We combine neuronal network simulations, mathematical analysis, and calcium imaging data to address the question of when and how functional connectivity, synaptic connectivity, and latent external input variability can be untangled. We show numerically and analytically that, even though the precision matrix of recorded spiking activity does not uniquely determine synaptic connectivity, it is often closely related to synaptic connectivity in practice under various network models. This relation becomes more pronounced when the spatial structure of neuronal variability is considered jointly with precision.

scholarly journals SynActJ: Easy-to-Use Automated Analysis of Synaptic Activity

2021 ◽  
Vol 3 ◽  
Author(s):  
Christopher Schmied ◽  
Tolga Soykan ◽  
Svenja Bolz ◽  
Volker Haucke ◽  
Martin Lehmann
Keyword(s):  
Image Analysis ◽  
Organic Dyes ◽  
Calcium Influx ◽  
Glutamate Release ◽  
Automated Analysis ◽  
Time Lapse ◽  
Synaptic Activity ◽  
Automated Image Analysis ◽  
Batch Mode ◽  
Fluorescent Intensity

Neuronal synapses are highly dynamic communication hubs that mediate chemical neurotransmission via the exocytic fusion and subsequent endocytic recycling of neurotransmitter-containing synaptic vesicles (SVs). Functional imaging tools allow for the direct visualization of synaptic activity by detecting action potentials, pre- or postsynaptic calcium influx, SV exo- and endocytosis, and glutamate release. Fluorescent organic dyes or synapse-targeted genetic molecular reporters, such as calcium, voltage or neurotransmitter sensors and synapto-pHluorins reveal synaptic activity by undergoing rapid changes in their fluorescence intensity upon neuronal activity on timescales of milliseconds to seconds, which typically are recorded by fast and sensitive widefield live cell microscopy. The analysis of the resulting time-lapse movies in the past has been performed by either manually picking individual structures, custom scripts that have not been made widely available to the scientific community, or advanced software toolboxes that are complicated to use. For the precise, unbiased and reproducible measurement of synaptic activity, it is key that the research community has access to bio-image analysis tools that are easy-to-apply and allow the automated detection of fluorescent intensity changes in active synapses. Here we present SynActJ (Synaptic Activity in ImageJ), an easy-to-use fully open-source workflow that enables automated image and data analysis of synaptic activity. The workflow consists of a Fiji plugin performing the automated image analysis of active synapses in time-lapse movies via an interactive seeded watershed segmentation that can be easily adjusted and applied to a dataset in batch mode. The extracted intensity traces of each synaptic bouton are automatically processed, analyzed, and plotted using an R Shiny workflow. We validate the workflow on time-lapse images of stimulated synapses expressing the SV exo-/endocytosis reporter Synaptophysin-pHluorin or a synapse-targeted calcium sensor, Synaptophysin-RGECO. We compare the automatic workflow to manual analysis and compute calcium-influx and SV exo-/endocytosis kinetics and other parameters for synaptic vesicle recycling under different conditions. We predict SynActJ to become an important tool for the analysis of synaptic activity and synapse properties.

scholarly journals Zebrafish Vascular Quantification (ZVQ): a tool for quantification of three-dimensional zebrafish cerebrovascular architecture by automated image analysis

Development ◽  
2022 ◽  
Author(s):  
E. C. Kugler ◽  
J. Frost ◽  
V. Silva ◽  
K. Plant ◽  
K. Chhabria ◽  
...  
Keyword(s):  
Image Analysis ◽  
Rapid Assessment ◽  
Three Dimensional ◽  
Vascular Anatomy ◽  
Light Sheet ◽  
Automated Image Analysis ◽  
Cerebral Vasculature ◽  
Experimental Conditions ◽  
Branching Points

Zebrafish transgenic lines and light sheet fluorescence microscopy allow in-depth insights into three-dimensional vascular development in vivo. However, quantification of the zebrafish cerebral vasculature in 3D remains highly challenging. Here, we describe and test an image analysis workflow for 3D quantification of the total or regional zebrafish brain vasculature, called zebrafish vasculature quantification “ZVQ”. It provides the first landmark- or object-based vascular inter-sample registration of the zebrafish cerebral vasculature, producing Population Average Maps allowing rapid assessment of intra- and inter-group vascular anatomy. ZVQ also extracts a range of quantitative vascular parameters from a user-specified Region of Interest including volume, surface area, density, branching points, length, radius, and complexity. Application of ZVQ to thirteen experimental conditions, including embryonic development, pharmacological manipulations and morpholino induced gene knockdown, shows ZVQ is robust, allows extraction of biologically relevant information and quantification of vascular alteration, and can provide novel insights into vascular biology. To allow dissemination, the code for quantification, a graphical user interface, and workflow documentation are provided. Together, ZVQ provides the first open-source quantitative approach to assess the 3D cerebrovascular architecture in zebrafish.

Cell interactions in the developing somite: in vitro comparisons between amputated (am/am) and normal mouse embryos

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 113-125
Author(s):  
O. P. Flint ◽  
D. A. Ede
Keyword(s):  
Limb Development ◽  
Mutant Mouse ◽  
Time Lapse ◽  
Cell Contact ◽  
Adhesive Properties ◽  
Cell Behaviour ◽  
Homozygous Mutant Mouse ◽  
Mutant Cells

Facial, axial and limb development are all abnormal in the homozygous mutant mouse embryo (amputated). An interpretation of cell behaviour in vivo based on sectioned material which may explain these abnormalities has been previously suggested. In this study, somite cells cultured in vitro were found to behave exactly as predicted in this interpretation: they clump together, forming extensive areas of cell contact, and this has a profound effect on their mobility as measured by time-lapse cinemicrography. The similarity of cell behaviour in vitro and in vivo under two distinct sets of environmental conditions suggests that the abnormal cell behaviour is intrinsic to the cell, and directly linked to the mutation. The more extensive areas of cell contact formed between mutant cells suggests that the mutation changes the adhesive properties of the cell surface, but it cannot be excluded that the cells' motile apparatus is also affected.

scholarly journals Age and prior exercise in vivo determine the subsequent in vitro molecular profile of myoblasts and nonmyogenic cells derived from human skeletal muscle

2019 ◽  
Vol 316 (6) ◽  
pp. C898-C912 ◽  
Author(s):  
Cecilie J. L. Bechshøft ◽  
Simon M. Jensen ◽  
Peter Schjerling ◽  
Jesper L. Andersen ◽  
Rene B. Svensson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Human Skeletal Muscle ◽  
Cell Function ◽  
Myogenic Differentiation ◽  
Molecular Profile ◽  
Muscle Stem Cell ◽  
Myogenic Cells

The decline in skeletal muscle regenerative capacity with age is partly attributed to muscle stem cell (satellite cell) dysfunction. Recent evidence has pointed to a strong interaction between myoblasts and fibroblasts, but the influence of age on this interaction is unknown. Additionally, while the native tissue environment is known to determine the properties of myogenic cells in vitro, how the aging process alters this cell memory has not been established at the molecular level. We recruited 12 young and 12 elderly women, who performed a single bout of heavy resistance exercise with the knee extensor muscles of one leg. Five days later, muscle biopsies were collected from both legs, and myogenic cells and nonmyogenic cells were isolated for in vitro experiments with mixed or separated cells and analyzed by immunostaining and RT-PCR. A lower myogenic fusion index was detected in the cells from the old versus young women, in association with differences in gene expression levels of key myogenic regulatory factors and senescence, which were further altered by performing exercise before tissue sampling. Coculture with nonmyogenic cells from the elderly led to a higher myogenic differentiation index compared with nonmyogenic cells from the young. These findings show that the in vitro phenotype and molecular profile of human skeletal muscle myoblasts and fibroblasts is determined by the age and exercise state of the original in vivo environment and help explain how exercise can enhance muscle stem cell function in old age.

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