We thank members of the Neumann lab for valuable discussions and input. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The authors thank WormBase for its wealth of information on C. elegans, and acknowledge Monash Micro Imaging, Monash University, for the provision of instrumentation, training and technical support. This work was supported by CMTAA research grants (2015 and 2018), and NHMRC Project Grants 1101974 and 1099690 awarded to B.N.

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The authors declare that they have no competing interests.

Morphological variations have frequently been assessed via manual counting of noticeable differences or using arbitrary thresholds to determine defects in comparison to a wild-type phenotype. More recently, however, quantitative methods have been used for comparative studies of morphology to accurately measure and describe changes on a cellular and subcellular level in an unbiased fashion. The ability to identify subtle yet biologically relevant differences between phenotypes is a powerful means for understanding the und...

The nematode Caenorhabditis elegans (C. elegans) is increasingly utilized as a model system to uncover the biological and molecular processes involved in human disease. An adult nematode has a body length of just over 1 mm, and can produce a large brood of up to 300 eggs1. After hatching, C. elegans only require 3-4 days to reach adulthood, and live for around 2 to 3 weeks2. Due to its ease of culturing, C. elegans is currently one of the most sought-after in vivo animal models for conducting cost-effective, rapid drug screening to identify therapeutics for human diseases. Additionally, its genetic conservation, well defined behavioral paradigms, transparent body for fluorescence or light microscopy, and ease of genetic manipulation make the study of cellular and molecular consequences of genetic mutations readily achieveable3. The C. elegans genome shares approximately 60-80% orthology with human genes, and about 40% of those genes are known to be disease-related. Some of human diseases that have been modelled and studied in C. elegans include neurodegenerative disorders (Alzheimer&#8217;s disease, Parkinson&#8217;s disease, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease), muscle-associated diseases (Duchenne muscular dystrophy), and metabolic diseases (hyperglycemia)2,4. In most human disorders, disease-induced cellular and organelle localization and morphological changes occur, which can readily be evaluated in the nematode model.
Fluorescent markers have been widely used to label tissues and organelles for dynamic visualization under the microscope. However, in C. elegans, conventional methods that assess morphological irregularities due to genetic mutations have largely relied on visual descriptions. While qualitative assessments can cover wider ranges of phenotypic descriptions (synaptic morphology, GFP clumping, specific axonal shape, muscle fiber thickness, etc.) and provide a bird&#8217;s eye view of the morphological changes, they are less well suited for comparing small variations across different groups. Furthermore, qualitative assessments are based on visual, subjective evaluation, which may lead to over- or under-estimations of morphological abnormalities. Finally, qualitative observations can also vary greatly between individuals, creating difficulties with data replication.&#160;
In recent years, a number of user-friendly, readily available computational algorithms that can quantitatively analyse images have been developed. However, the utilization of such image analysis software for some morphological studies, especially in relation to body wall muscles and mitochondria, in C. elegans research has lagged behind. To improve underlying structural analysis in C. elegans, some of the readily available, open-source image analysis software were trialed to quantitatively compare the effects of genetic mutations on muscle mitochondria, body wall muscle and synaptic morphology. These experimental procedures outline in detail how these programs (Fiji, ilastik, CellProfiler, SQUASSH) can be used to evaluate changes in synaptic size and synaptic protein localization, body wall muscle area and fiber length, and mitochondrial size and circularity as a result of genetic mutations in the nematode.&#160;

<table><tbody><tr><td>Agar-agar</td><td>Merck</td><td>1.01614.1000</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP8</td><td>Inverted platform</td></tr><tr><td>Fluorescence microscope</td><td>Carl Zeiss AG</td><td>Zeiss Axio Imager M2</td><td></td></tr><tr><td>Glass coverslips #1</td><td>Thermo scientifique</td><td>MENCS22221GP</td><td></td></tr><tr><td>Glass coverslips #1.5</td><td>Zeiss</td><td>474030-9000-000</td><td>Made by SCHOTT</td></tr><tr><td>Glass slides</td><td>Thermo scientifique</td><td>MENS41104A/40</td><td></td></tr><tr><td>Light LED</td><td>Schott</td><td>KL 300 LED</td><td></td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Tryptone (Peptone from casein)</td><td>Merck</td><td>107213</td><td>Ingredients for Lysogeny Broth (LB) medium</td></tr><tr><td>Yeast Extract</td><td>Merck</td><td>103753</td><td>Ingredients for Lysogeny Broth (LB) medium</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>106404</td><td>Ingredients for Lysogeny Broth (LB) medium</td></tr><tr><td>Peptone (Peptone from meat)</td><td>Merck</td><td>107214</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>106404</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8667-25G</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102382</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Magnesium sulfate</td><td>Merck</td><td>105886</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Dipotassium phosphate</td><td>Merck</td><td>105101</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>104873</td><td>Ingredients for Nematode Growth Media (NGM) agar</td></tr><tr><td>Disodium phosphate</td><td>Merck</td><td>106586</td><td>Ingredients for M9 buffer</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>106404</td><td>Ingredients for M9 buffer</td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>104873</td><td>Ingredients for M9 buffer</td></tr><tr><td>Magnesium sulfate</td><td>Merck</td><td>105886</td><td>Ingredients for M9 buffer</td></tr><tr><td>Pasteur pipette</td><td>Corning</td><td>CLS7095D5X-200EA</td><td></td></tr><tr><td>Petri dishes</td><td>Corning</td><td>CLS430589-500EA</td><td></td></tr><tr><td>Platinum wire</td><td>Sigma</td><td>267201-2G</td><td></td></tr><tr><td>Spatula</td><td>Met-app</td><td>2616</td><td></td></tr><tr><td>Tetramisole hydrochloride</td><td>Sigma</td><td>L9756-5G</td><td></td></tr></tbody></table>

quantitative approaches for studying cellular structures and organelle morphology in caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

C. elegans is an ideal model organism for studying morphology of different tissues and organelles due to its simplicity, known cell lineage, transparency, and available tools. Here, we provide quantitative approaches for studying organelles (e.g., mitochondria) and tissues, including synapses and muscles using live fluorescence imaging and free bio-image processing software.
Strict regulation of MEC-17 expressio...

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Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

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9.6K Views.  Monash University, Melbourne VIC 3800, Australia.. Het vermogen om veranderingen in de morfologie van weefsels en organellen te kwantificeren is belangrijk voor het begrijpen van de genfunctie en de impact van genetische mutaties. Onze protocollen leggen uit hoe vrij beschikbare software kan worden gebruikt voor niet-subjectief beoordelen van de morfologie van synapsen, spieren en mitochondriën in een nematode, C.elegans. Veel eerdere studies hebben vertrouwd op kwalitatieve methoden voor het vergelijken van morfologische veranderingen.

Video: Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Developmental Biology

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Behavior

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Neuroscience

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioengineering

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

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