The authors have nothing to disclose.

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Research in the lab of CP is funded by the Deutsche Forschungsgemeinschaft (EXC 115; FOR 1756) and the LOEWE Research Cluster Ubiquitin Networks. CP is further supported by the European Union Framework Program 7 (Marie Curie Actions Project 326632).

באמצעות מעקב אחר אובייקט בפיתוח, במעקב גרעיני מסוים, זה כבר אפשרי להבהיר מנגנוני דפוסים מרכזיים של ג elegans עובר 1,23,24. הרחבת אסטרטגיה זו לתפוקה גבוהה יותר, זה כבר אפשרי לאחרונה לחשוף כללי דפוסים נוספים ולהציע שיטה איך להסיק דפוסים שולטת דה נובו 10. ל?...

עם השיפורים מתמידים של חלבוני ניאון, הנדסה גנטית, מיקרוסקופ אור, ורך מחשב וחומרה, עכשיו זה אפשרי להקליט פיתוח של אורגניזמים מודל רבים ברזולוציה מרחב ובזמן חסרת תקדים. הדבר מאפשר לחוקרים לשאול שאלות שלא ניתן לטפל בעבר או לבקר תהליכים התפתחותיים ידועים כדי לחפש את היבטים התעלמו. התקדמות זו עוררה את תחום הביולוגיה התפתחותית כמותיים, שמטרתה להפוך את הדגמים איכותיים, פורמליים למודלים כמותיים על ידי מדידות יסודיות וניתוחים סטטיסטיים. תאי מעקב ומבני subcellular הפכו אותו ניתן להפיק מודלים כמותיים של התפתחות עוברית, פעילות מערכת עצבים, או תא חלוקה 1-12. על ידי מעקב אחר שרידי חלוקת התא במהלך ההתפתחות המוקדמת של ג עובר elegans, אנו יכולים לחשוף לאחרונה שהם בצעו סטריאוקיטוב חשוב הקליד נתיב ומהווה גורמי 13,14. הנה, פרוטוקולים מוצגים שהופכים את הביולוגיה התפתחותית כמותיים גישות נגישות לאינם מומחים. המוקד נמצא על שלושה כלים ישר קדימה, זמינים באופן חופשי כי הם ישימים בכל מעבדה יש ​​לו גישה למיקרוסקופיה confocal סטנדרטית ומחשבים. אלה כוללים פרוטוקול כדי ליצור צורות תא 3D, פרוטוקול כדי לעקוב אחר שרידי חלוקת תא, ופרוטוקול לתאר כמותית דינמיקת actomyosin קליפת המוח. נמטודות ג elegans משמש כמקרה למופת, לעומת זאת, השיטות וכלים שנדונו כאן הם מתאימות למגוון רחב של שאלות במודלים אחרים ביולוגיים, למשל, תאים בתרבית, explants רקמות, organoids או spheroids, עוברים אחרים, וכו &#39; בדרך כלל, חלק מהניתוחים המוצגים כאן יכול גם להתבצע עם ImageJ כלי הקוד פתוח הפופולרי (http://imagej.nih.gov/ij/docs/index.html; או פיג&#39;י, &quot;הסוללות כלולות&quot; גרסה של ImageJ, http://fiji.sc/Fiji) שלתוספים רבים לניתוחים כמותיים שונים זמינים. עם זאת, התוכניות שנדונו כאן נועדו להתמודד עם בעיות ספציפיות. ראשית, IMOD, עיבוד תמונה, דוגמנות וחבילת תצוגה יכולים לשמש לשחזורי 3D סעיפים סדרתי מאלקטרון או מיקרוסקופ אור 15. IMOD מכיל גם כלים להצגת נתוני 3D מכל כיוון. שנית, Endrov, תכנית Java נועדה לבצע ניתוח תמונה, עיבוד נתונים, וביאור של רשתות או רצועות (בין יתר) על בסיס ארכיטקטורת תוסף מורחב, עם תאימות תוסף ImageJ 16. הוא מכיל מעל 140 פעולות עיבוד תמונה וממשק משתמש להרחבה שבמודל והנתונים גולמיים מוצגים בנפרד. ניתן למצוא את קוד המקור שלה בhttps://github.com/mahogny/Endrov. שלישית, PIVlab, Mכלי ATLAB לvelocimetry תמונת חלקיק דיגיטלי המאפשר למשתמש כמותית ואיכותי לנתח זרימת חלקיקי שדות 17. השימוש בתוכניות זה טעון רישוי MATLAB הכולל עיבוד תמונת ארגז כלים (http://mathworks.com). PIVlab היא תכנית שנועדה לתאר זרימת כמותית. היא מחשבת את חלוקת מהירות, גודל, ערבוליות, סטייה, או הגזירה בתוך זוגות תמונה או סדרה. לשם כך, בין-קורלציה אזורים קטנים של תמונות (&#39;עובר&#39; בשם בסעיף הפרוטוקול) של צמד תמונה לגזור את עקירת חלקיקים הסבירה ביותר. חוצה קשר זה מניב מטריצת מתאם שניתן לנתח בתחום החלל או תדירות או באמצעות מתאם צולב ישיר או שינוי פורייה מהיר (FFT), בהתאמה. הציוד המשמש כאן הוא מיקרוסקופ הפוך מצויד בניפקוב (&quot;ספינינג&quot;) דיסק, EMCCD, 488 ומוצק 561 ננומטר סטנדרטילייזרים של מצב, ו20x אוויר או 40x או 60x תכנית / Apochromat מטרות נפט או מים טבילה. עם זאת, אפשר גם לבצע הדמיה הזמן לשגות עם שיטות הדמיה אחרות, למשל, על נקודה, סכן למעשה או מיקרוסקופיה גיליונות סריקה מבוססת לייזר, מיקרוסקופיה רב-פוטון, כמו גם במיקרוסקופ פלואורסצנטי עלית בשילוב עם deconvolution או מובנה תְאוּרָה. היתרון של שימוש במערכת דיסק ניפקוב הוא רכישת תמונה מאוד מהר, במיוחד אם מצב הזרמה (תנועה וסריקה של האובייקט בממד z רציף) זמין. בנוסף, כדי לשפר את הרזולוציה, ניתן להשתמש במאריך מגדלת פי 1.5 מול EMCCD.

<table><tbody><tr><td>Stereo microscope</td><td>Motic/VWR</td><td>OT4005S</td><td>Stereo microscope for dissection and mounting</td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>18329</td><td>Embryo mounting</td></tr><tr><td>20 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Polybead Polystyrene Microspheres,&amp;nbsp;</td><td>Polysciences</td><td>876</td><td>Adult animal mounting</td></tr><tr><td>0.1 &amp;micro;m</td><td></td><td></td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>631-0902</td><td>Adult animal mounting</td></tr><tr><td>Cover glass 18x18 mm</td><td>VWR</td><td>631-1331</td><td>Embryo/adult mounting</td></tr><tr><td>Cover glass 24x60 mm</td><td>VWR</td><td>631-1339</td><td>Embryo mounting</td></tr><tr><td>Scalpel</td><td>VWR</td><td>233-5455</td><td>Embryo dissection</td></tr><tr><td>Silicone tubing</td><td>VWR</td><td>228-1501</td><td>Tubing for mouth pipette</td></tr><tr><td>30 mm PTFE membrane filter</td><td>NeoLab</td><td>Jul-01</td><td>Filter for mouth pipette</td></tr><tr><td>Capillary tubes</td><td>VWR</td><td>621-0003</td><td>Pipette tip for mouth pipette</td></tr><tr><td>Vaseline</td><td>Roth</td><td>E746.1</td><td>Embryo/adult mounting</td></tr><tr><td>Agar</td><td>Roth</td><td>5210.5</td><td>Adult animal mounting</td></tr><tr><td>Potassium-di-hydrogenphosphate</td><td>Roth</td><td>P018.2</td><td>M9 buffer</td></tr><tr><td>Di-sodium- hydrogenphosphate</td><td>Roth</td><td>P030.2</td><td>M9 buffer</td></tr><tr><td>Sodium chloride</td><td>Roth</td><td>3957.1</td><td>M9 buffer</td></tr><tr><td>VisiScope Spinning Disc Confocal System</td><td>Visitron Systems</td><td>n/a</td><td>Confocal microscopy</td></tr></tbody></table>

tracking and quantifying developmental processes in c. elegans using open-source tools

כמותית לכידת תהליכים התפתחותיים היא חיונית כדי להפיק מודלים ומפתח מכניסטית לזהות ולתאר פנוטיפים מוטציה. כאן פרוטוקולים מוצגים להכנת עוברים וג הבוגר elegans בעלי חיים למיקרוסקופיה זמן לשגות טווח קצר וארוך ושיטות למעקב וכימות של תהליכים התפתחותיים. כל השיטות שהוצגו מבוססות על ג זנים זמינים במרכז לגנטיקת Caenorhabditis ועל תוכנת קוד פתוח elegans שניתן ליישם בקלות בכל מעבדה עצמאית של מערכת מיקרוסקופיה בשימוש. בנייה מחדש של מודל תא-צורת 3D באמצעות IMOD תוכנת דוגמנות, (velocimetry תמונה דיגיטלי חלקיקים זמן נפתר מעקב ידני של מבני subcellular fluorescently שכותרתו באמצעות התכנית רב תכליתי ניתוח תמונת Endrov, וניתוח של זרימת התכווצות בקליפת המוח באמצעות PIVlab כלי לMATLAB) מוצג. הוא דן כיצד גם ניתן לפרוס שיטות אלה לאo ללכוד כמותית תהליכים התפתחותיים אחרים בדגמים שונים, לדוגמא, מעקב תא ושושלת ההתחקות, מעקב של זרימת שלפוחית.

על ידי שימוש בפרוטוקולים 2, 3, ו -4, הזמן לשגות הדמיה של בלוטות מין בג הסוג בר elegans מבוגרים מתבצע (OD58 מתח (UNC-119 () III ed3; ltIs38 [pAA1; עוגה-1 :: GFP :: PH (PLC1delta1) + UNC-119 (+)]), מבטא את סמן קרום מאמרגן germline ). התמקדות בתור של בלוטת המין, מודל 3D של תאי הנבט נוצר מנתונים מיקרוסקופ (איו?...

Watch this Scientific Journal Video about Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools at JoVE.com

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

מעקב וכימות תהליכים התפתחותיים ב<em&gt; C. elegans</em&gt; שימוש בכלי קוד פתוח

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 ...

tracking-quantifying-developmental-processes-c-elegans-using-open

Research

JoVE Journal

Biology

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

8.8K Views.  Goethe University. 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.

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

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis מבוסס microfluidic לניתוח כמותי על פי דרישה של<em&gt; Elegans Caenorhabditis</em&gt; &#39;נידות

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Bioengineering

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

מעקב סי אלגנס ומדידת התנהגות

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

Neuroscience

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.

איזוטרופי מיקרוסקופ גיליון אור אוטומטי שושלת היוחסין ניתוח לקטלוג Caenorditis הEmbryogenesis עם רזולוציה Subcellular

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

Developmental Biology

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.

ניתוח השוואתי של שיטות ניסיוניות לכימות פעילות בעלי חיים בקאנורבדיטיס אלגנס מודלים של מחלות מיטוכונדריאליות

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

Behavior

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

שיטת דימות למחצה של תפוקה גבוהה וערכת כלי הדמיה של נתונים כדי לנתח את הפיתוח העובריים של ס. אלאנס

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

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.

Microchambers agarose לסידן הדמיה לטווח ארוך של<em&gt; Elegans Caenorhabditis</em

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

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.

הדמיה ג elegans עוברים באמצעות מיקרוסקופ Epifluorescent ותוכנות קוד פתוח

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

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

ניתוח אוטומטי של<em&gt; C. elegans</em&gt; התנהגות שח שימוש CeleST תוכנה

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

אוטומטיות התנהגותית ניתוח של גדול C. elegans אוכלוסיות שימוש רחב שדה-of-view מעקב פלטפורמה

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

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.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

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

מעקב וכימות תהליכים התפתחותיים ב

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Electrotaxis מבוסס microfluidic לניתוח כמותי על פי דרישה של

מעקב סי אלגנס ומדידת התנהגות

איזוטרופי מיקרוסקופ גיליון אור אוטומטי שושלת היוחסין ניתוח לקטלוג Caenorditis הEmbryogenesis עם רזולוציה Subcellular

ניתוח השוואתי של שיטות ניסיוניות לכימות פעילות בעלי חיים בקאנורבדיטיס אלגנס מודלים של מחלות מיטוכונדריאליות

שיטת דימות למחצה של תפוקה גבוהה וערכת כלי הדמיה של נתונים כדי לנתח את הפיתוח העובריים של ס. אלאנס

Microchambers agarose לסידן הדמיה לטווח ארוך של

הדמיה ג elegans עוברים באמצעות מיקרוסקופ Epifluorescent ותוכנות קוד פתוח

ניתוח אוטומטי של

אוטומטיות התנהגותית ניתוח של גדול C. elegans אוכלוסיות שימוש רחב שדה-of-view מעקב פלטפורמה

בסיסי