The authors have nothing to disclose.

1. Preparation of C. elegans Embryos for Time-lapse Microscopy using Microbeads
<ol>
	<li>Transfer gravid adult worms by using a worm pick (platinum wire) into a drop of M9 buffer and clean off bacteria by first vigorously agitating and then transferring each worm to an adjacent drop of M9 using an eye lash mounted onto a tooth pick/pipette tip or use a pointed glass capillary.</li>
	<li>Dilute the dispersion containing the 20 &#181;m diameter polystyrene beads 10-fold in M9 buffer (1 L M9 buffer contains 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, and, after autoclaving (20 min, 120 &#176;C), add an additional 1 ml of a 1 M MgSO4 solution).</li>
	<li>Make a mouth pipette from any type of thin glass capillary (e.g., melting point capillaries or capillaries used to pull injection needles), a roughly 0.5 m long piece of rubber, silicone or similar material tubing (ideally transparent), a 200 &#181;l pipette tip, the lid of a 200 &#181;l PCR tube, a hydrophobic filter for small syringes (e.g., a 0.2-0.45 &#181;m pore size and 15-50 mm diameter Nylon, PTFE or similar material filter), and a 1000 &#181;l pipette tip. This type of mouth pipette ensures that no biological material can get in contact with the experimenter.</li>
	<li>Assemble the pipette by inserting the 200 &#181;l pipette tip with the tip first into the tubing and plug it with the lid of a PCR tube which has a central perforation that matches the diameter of the capillary used. A very pointed awl or preparation needle can be used to generate the perforation.</li>
	<li>Draw a thin capillary and insert it into the perforation. Insert the filter into the other end of the tubing and insert a 1000 &#181;l pipette tip as mouth piece (Figure 1).</li>
	<li>Dissect gravid adults by cutting worms transversely at the vulva with a clean razor blade or scalpel. 
	NOTE: Usually, many embryos will be expulsed after cutting. However, if early embryos are needed, they sometimes have to be released from the carcass using an eye lash or pointed needle.</li>
	<li>Pipette one drop of the diluted polystyrene bead dispersion (~4 &#181;l) onto a 24x60 mm cover glass and transfer embryos into this drop using the mouth pipette.</li>
	<li>Carefully place a small (e.g.,&#160;18x18 or 20x20 mm) cover glass on top of the drop and make sure the drop has fully spread out. 
	NOTE: The drop should also be small enough not to touch the edges of the small cover glass.</li>
	<li>Seal the mount with liquefied vaseline. Add a drop of immersion oil on the 24x60 mm cover slip side and proceed to the &#39;Time-lapse Microscopy&#39; section.</li>
</ol>
2. Preparation of Adult C. elegans Animals for Time-lapse Microscopy using Nanobeads
NOTE: In general, the protocol for mounting adult animals is an adaptation of the protocol from ref. 18. With this protocol, it is possible to perform long-term time-lapse confocal microscopy of adult animals.
<ol>
	<li>Prepare a 1.5% (w/v) agarose solution in M9 buffer, boil (1 min, 100 &#176;C in water bath or microwave). The use of higher concentrations of agarose is not recommended since this can lead to extrusion of the intestine and/or parts of the gonad.</li>
	<li>Stick down 2-3 layers of tape onto two microscope slides each. Place a slide without tape between the two slides with tape.</li>
	<li>Use a pipette tip and place one drop of the hot agarose solution in the center of the middle slide. Quickly place another slide onto the drop so that it is supported by the tape layers on each side. This will create a round and flat agar pad of around 10 mm diameter.</li>
	<li>Put a drop of 100 nm bead solution (undiluted, which is 2.6% (w/v)) onto the pad. Transfer 1-10 cleaned adult worms into the drop with a worm pick. Seal with 18x18 mm cover glass and vaseline as explained in Protocol 1.</li>
</ol>
3. Time-lapse microscopy
<ol>
	<li>Put a drop of immersion oil onto the microscope slide or the cover glass sandwich. Use brightfield at low magnification (5x or 10x lenses) to locate the sample. 
	NOTE: Limit the exposure to blue light as much as possible (in particular for C. elegans embryos), e.g., do not use epi-fluorescence microscopy to find/focus the specimen but use brightfield instead.</li>
	<li>Change to higher magnification (40x or 60x objectives), bring the sample into focus (either central plane or top/bottom planes of the sample, depending on the acquisition software).</li>
	<li>Set the sampling intervals to 30 planes at 1 &#181;m spacing recorded every 1min. In case an automated xy- or xyz-stage is available, multiple spots can be recorded in one session. Switch to fluorescence imaging and check the focus again. 
	NOTE: Try to avoid using high laser powers, rather use slightly longer exposure times at very low intensities. Measure acquired images right away, either in the image acquisition program or in ImageJ to avoid saturation, which crops the dynamics range and also means excessive radiation exposure.</li>
	<li>Start recording the time-lapse series. 
	NOTE: To exclude analyzing artifacts, start with long (3-10 min) time intervals between sample scanning and gradually increase temporal sampling to the desired intervals. Developmental timing as well as end points in both cases should be similar when performing a statistical analysis. Moreover, it is often necessary to assess phototoxicity and survival rates. Therefore, recover specimen from the mount after long-term time-lapse microscopy and check whether they continue to develop/live.</li>
</ol>
4. Reconstructing a 3D Cell Shape model with IMOD
NOTE: IMOD software is available from the IMOD web page: http://bio3d.colorado.edu/imod/. Installation of the software is described there. When using Windows OS, a Unix toolkit has to be installed, which can also be found as a package on the IMOD webpage. Moreover, there is an excellently compiled 3dmod introduction available on the IMOD webpage (http://bio3d.colorado.edu/imod/doc/3dmodguide.html).
<ol>
	<li>Open the Unix shell and type in &#34;imod&#34;. The IMOD window will open. Select the file with the raw data (ideally a stacked tif or similar) from which to generate a 3D model.</li>
	<li>Locate the bottom and top of the objects in the ZaP window by using the information window (up&#38;down scrolling tool).</li>
	<li>Start tracing the cell outlines. Importantly, ensure that each cell is one object. Begin with the top plane of each cell and create the first contour there using the information window. Scroll down in z and create a new contour for each z-plane.</li>
	<li>Do the same for as many cells as needed to model in 3D.&#160; Do not forget to create a new object when starting with a new cell. 
	NOTE: There are many different ways for contour tracing and several helpful plugins. Please visit the IMOD webpage and online tutorials for details. In the case shown here, the default setting has been used.</li>
	<li>Open the &#34;Model View&#34; window to inspect the objects and their contours.</li>
	<li>In the &#34;Model View&#34; toolbar choose &#34;Edit&#34; -&#34;Objects&#34;. The respective editing window will open.</li>
	<li>To model cells as filled and closed objects, choose &#34;Mesh&#34; as &#34;Draw Data Type&#34; and &#34;Fill&#34; as &#34;Drawing Style&#34;. Click on &#34;Meshing&#34; in the scrollable selection at the lower left of the window. Check the option &#34;Cap&#34; in the selection at the lower right. Check as many objects as necessary and click &#34;Mesh All&#34;. The program will generate solid objects from the stacks of contours. The z-sampling factor can be adjusted by changing &#34;Z-scale&#34; in the &#34;View&#34; window.</li>
	<li>Move/rotate the 3D object (&#34;Edit&#34; - &#34;View&#34;) and save snapshots or movies (&#34;File&#34; -&#34;Snap Tiff As ...&#34; or &#34;Movie/Montage&#34;).</li>
</ol>
5. Tracking Fluorescently Labeled Structures with Endrov
NOTE: In order to track cell division remnants, use a strain with a nuclear or plasma membrane marker for cell identification (e.g., H2B, PH-PLC-d1) and a cell division remnant marker (e.g., NMY-2, ZEN-4). The nuclear or plasma membrane marker is important to determine which cell inherits the cell division remnant.
<ol>
	<li>For fast loading and ease of use, the raw data has to be converted to a tiff file (e.g., in ImageJ). Next, open Endrov and load the file to be analysed by choosing the &#34;Load file&#34; option from the file menu. Click on the &#34;2D viewer&#34; icon on the toolbar.</li>
	<li>Adjust the histogram by clicking on the &#34;Fit range&#34; icon (the blue icon at the bottom right corner of the Endrov screen). Set the XYZ-resolution by going to &#34;Data&#34; -&#34;filename&#34;-&#34;Imageset&#34; -&#34;Channel&#34;-&#34;Set XYZ-resolution&#34;. Type in values for X, Y, and Z, e.g., 5, 5, 1 if X and Y resolution is 0.2 &#181;m and sample every 1 &#181;m in Z.</li>
	<li>For annotating the nuclei, move to the plane of interest and click on &#34;2D viewer&#34;. Select &#34;Lineage&#34; from the dropdown menu and choose &#34;New lineage&#34;.</li>
	<li>By clicking and dragging on the nucleus, it can be marked. To name the nucleus, right click and choose &#34;Rename particle&#34; from the dropdown menu. To increase or decrease the diameter of the circle, drag the arrow icon that appears on the left of the marked circle. To mark the central plane of the nucleus, click on the right icon. When using a plasma membrane marker, a large sphere can be drawn that will cover most of the cell.</li>
	<li>To proceed in time and plane, choose the &#34;Frame&#34; and &#34;Z&#34; options at the bottom toolbar. After going to the next time point, adjust the position or size of the circle that marks the nucleus accordingly. Repeat this until the tracked cell divides.</li>
	<li>When there is a cell division, mark the daughter nuclei and switch to &#34;Lineage viewer&#34;. This can be selected by clicking on the &#34;Windows&#34; icon on the top toolbar. Mark all three nuclei simultaneously, go to the &#34;Lineage&#34; option in the top toolbar and select &#34;Associate parent&#34;.</li>
	<li>When cell tracking is finished, switch to the channel marking the cell division remnant for tracking. To switch channels, click on the file name on the bottom left corner of the toolbar. The cell division remnant (or any other structure) can be tracked as described above for the nucleus.</li>
	<li>When returning to the software after closing, it is necessary to go to &#34;2D Viewer&#34; -&#34;Lineage&#34;-&#34;Edit&#34; and to click on the lineage number that will be edited.</li>
</ol>
6. Analyzing Cortical Actomyosin Flow with PIVlab
NOTE: PIVlab is a freely available software from: &#160;http://pivlab.blogspot.de/; it can be invoked from within the Matlab command line environment by typing PIVlab_GUI. When working with PIVlab, it is recommended to save the image series in a separate folder.
<ol>
	<li>Start a new session from &#34;File menu&#34;. Load the image files by clicking the &#34;Load images&#34; button.</li>
	<li>Select the sequencing style 1-2, 2-3, ... and navigate to the directory that contains the sequence of desired images. Select the images and click on the &#34;Add&#34; button and import.</li>
	<li>Go to &#34;Analyses settings&#34; and select &#34;Exclusions (ROI,Mask)&#34;. Alternatively, press &#34;Ctrl + E&#34;. Use the button &#34;Draw mask(s) for current frame&#34; to inactivate the undesired part of the image/s (i.e., area outside embryo).</li>
	<li>Select &#34;PIV settings&#34; from the &#34;Analyses settings&#34; menu. Alternatively, press &#34;Ctrl + S&#34;. Choose &#34;FFT window deformation&#34; as the desired PIV algorithm. 
	NOTE: Fast Fourier transformation (FFT) reduces the computational cost but is only recommended the signal is well above noise and if the displacement of the particles studied does not exceed half the pass area analyzed (see 6.5).</li>
	<li>Enter the following values for the three passes, which are the area of interrogation for intensity cross correlation between subsequent images: Pass 1: Interrogation area of 64 pixels with a step of 32. Pass 2: Interrogation area of 32 pixels with a step of 16. Select &#34;linear&#34; from the Window deformation drop down menu. Choose Gaussian 2*3-point method for sub-pixel displacement estimation.</li>
	<li>Select &#34;Analyze!&#34; from Analysis menu and use the button &#34;Analyze all frames&#34; to begin an analysis.</li>
	<li>For post processing select &#34;Vector validation&#34; from &#34;Post processing&#34; menu and set the standard deviation (Stdev) filter threshold to 7 and apply to all frames.</li>
	<li>Choose &#34;Derive parameters/ modify data&#34; from &#34;Plot&#34; menu. Select &#34;Vectors [px/frame]&#34; from dropdown menu. Adjust the smoothness of vectors and high pass filter and apply to all frames.</li>
	<li>To calculate mean vectors of all frames, set the &#34;Used frames&#34; to 1:end and click on &#34;Calculate mean vectors&#34; button.</li>
</ol>

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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).

Through object tracking in development, in particular nuclear tracking, it has been possible to elucidate central patterning mechanisms of C. elegans embryogenesis1,23,24. Expanding this strategy to higher throughput, it has been recently possible to uncover additional patterning rules and to propose a method how to deduce patterning rules de novo10. For many mutants, however, the precise patterning defects are still unknown. The methods described here are tools that can be instrum...

With the steady improvements of fluorescent proteins, genome engineering, light microscopy, and computer soft- and hardware, it is now possible to record development of many model organisms at unprecedented spatio-temporal resolution. This allows researchers to ask questions that could not be addressed previously or to revisit known developmental processes in order to search for overlooked aspects. This progress has sparked the field of quantitative developmental biology, which aims at transforming qualitative, informal models into quantitative models by thorough measurements and statistical analyses.
Tracking cells and subcellular structures has made it possible to derive quantitative models of embryonic development, nervous system activity, or cell division1-12. By tracking cell division remnants during early development of the C. elegans embryo, we could recently reveal that they follow a stereotyped path and constitute important polarizing factors13,14.
Here, protocols are presented that make quantitative developmental biology approaches accessible for non-experts. The focus lies on three straight-forward, freely available tools that are implementable in any lab that has access to standard confocal microscopy and computers. These include a protocol to generate 3D cell shapes, a protocol to track cell division remnants, and a protocol to quantitatively describe cortical actomyosin dynamics. The nematode C. elegans is used as an exemplary case, however, the methods and tools discussed here are suited for a variety of questions in other biological models, e.g., cultured cells, tissue explants, organoids or spheroids, other embryos, etc.
Generally, some of the analyses shown here can also be performed with the popular open source tool ImageJ (http://imagej.nih.gov/ij/docs/index.html; or FIJI, the &#8216;batteries included&#8217; version of ImageJ, http://fiji.sc/Fiji) for which many plugins for different quantitative analyses are available. However, the programs discussed here are designed to tackle specific problems.
Firstly, IMOD, an image processing, modeling and display suite can be used for 3D reconstructions of serial sections from electron or light microscopy15. IMOD also contains tools for viewing the 3D data from any orientation. Secondly, Endrov, a Java program designed to perform image analysis, data processing, and annotation of networks or tracks (among others) on the basis of an extended plugin architecture, with ImageJ plugin compatibility16. It contains over 140 image-processing operations and an extensible user interface in which model and raw data are displayed separately. Its source code can be found at https://github.com/mahogny/Endrov. Thirdly, PIVlab, a MATLAB tool for digital particle image velocimetry that allows the user to quantitatively and qualitatively analyze particle flow fields17. The use of this programs requires a MATLAB license that includes the Image Processing Toolbox (http://mathworks.com). PIVlab is a program designed to quantitatively describe flow. It calculates the velocity distribution, magnitude, vorticity, divergence, or shear within image pairs or series. For this, it cross-correlates small areas of images (called &#8216;passes&#8217; in the protocol section) of an image pair to derive the most probable particle displacement. This cross-correlation yields a correlation matrix that can be analyzed in the space or frequency domain using either direct cross-correlation or a fast Fourier transformation (FFT), respectively.
The equipment used here is an inverted microscope equipped with a Nipkow (&#39;spinning&#39;) disc, an EMCCD, 488 and 561 nm standard solid-state lasers, and 20x air or 40x or 60x plan/apochromat oil- or water-immersion objectives. However, it is also possible to perform time-lapse imaging with other imaging modalities, e.g., point-, line- or sheet-scanning laser-based microscopy, multi-photon microscopy, as well as epi-fluorescence microscopy combined with deconvolution or structured illumination. The advantage of using a Nipkow disc system is the extremely fast image acquisition, especially if a streaming mode (continuous movement and scanning of the object in the z dimension) is available. In addition, to improve resolution, a 1.5-fold magnifying extender in front of the EMCCD can be used.

<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

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.

By using protocols 2, 3, and 4, time-lapse imaging of gonads in wild type C. elegans adults is performed (strain OD58 (unc-119(ed3) III; ltIs38[pAA1; pie-1::GFP::PH(PLC1delta1) + unc-119(+)]), expressing a membrane marker from a germline promoter). Focusing on the turn of the gonad, a 3D model of the germ cells is generated from the microscopy data (Figure 2). This model allows to analyze changes in cell size while the cells transit form the distal to the proximal arm, reveals the organization o...

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

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.

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 ...

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Research

JoVE Journal

Biology

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

Summary

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Chapters in this video

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

C. elegans Tracking and Behavioral Measurement

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

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

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

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

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

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation