We thank CIFF imaging facility, NCBS for use of the confocal microscopes supported by the DST - Centre for Nanotechnology (No. SR/55/NM-36-2005). We thank research funding from DBT (SPK), CSIR-UGC (JD), DST (SM), DBT (SM), spinning disc supported by DAE-PRISM 12-R&#38;D-IMS-5.02.0202 (SPK and Gautam Menon), and HHMI-IECS grant number 55007425 (SPK). HB101, PS3239, and wdIs51 strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). S.P.K. made jsIs609 in Mike Nonet&#39;s Laboratory.

<ol>
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S.M. and S.P.K. are authors of a pending patent on the microfluidic growth and imaging device (Patent application number 640/CHE/2011).

In this paper, a protocol for fabrication and use of a simple microfluidic device for growing C. elegans with constant food supply and high-resolution imaging of a single animal during its development has been described. This fabrication process is simple and can be done in a non-sterile environment. A dust-free environment is critical during fabrication steps. The presence of dust particles would lead to improper contact between the two bonding surfaces, resulting in poor bonding and leakage of the device during high-pressure application while C. elegans are immobilized. Out of all the devices fabricated, 95% of the devices are suitable for experiments. A small number of devices fail (5%) due to inappropriate bonding during fabrication or use. Failure in bonding can be reduced by carrying out the fabrication process inside a dust-free (&gt; 1,000-grade clean room) available at several academic institutions around the globe. To clean bacterial food from the flow channel and enable device reuse, flush the device by flowing alcohol through it after every experiment. 
The protocol demonstrates a lithography fabrication process with a simple design that can easily be modified for different developmental stages and sizes of C. elegans. Further, this device design can be adapted for other model organisms like zebrafish and Drosophila, by increasing the dimensions of the flow and trapping layers3 to observe a variety of developmental and sub-cellular processes. In this protocol, two different heights of the flow layer channel - 40 &#181;m and 80 &#181;m - have been used for complete immobilization and tracking of cellular and sub-cellular features in different developmental stages of C. elegans as appropriate for their body sizes. For example, the dendritic arborization in the PVD sensory neurons begins in the early L2 stages and continues up to the L4 stage. This was imaged in a device with a 40 &#181;m high flow layer. As the PVD neurons form dendrites that innervate body wall muscles it requires optical sectioning to quantify the processes in 3D. The quantitative analysis of dendritic arbors requires complete immobilization of the animals within the device that matches the body diameter. However, for monitoring vulval development, 80 &#181;m height of flow layer was used to track the vulval lineages. For TRNs length and mitochondria imaging, we imaged L2 to L4 stages using devices with 40 &#181;m flow layer thickness. Using a device with the wrong height will cause difficulty in immobilization. For example, small animals (L2 or early L3 stages) inside an 80 &#181;m flow layer height device will permit animal flipping inside the device and show incomplete immobilization even after the trapping membrane is under 14 psi pressure. On the other hand, animals with large body diameters (beyond late L4) do not easily enter inside a device with 40 &#181;m flow layer height. Trapping large animals in small devices under high pressure can damage their body. The application of the current device with a 40 &#181;m flow layer is limited and not suitable for high-resolution imaging of very young early larval stage animals (such as L1 stage animals younger than 10 h after hatching) as they are not completely immobilized under the membrane. Due to small body sizes, the small size larval animals keep moving and occasionally escape the trap when they are excited with laser light. For L1 stage animals, one can perform low-resolution bright field or fluorescence imaging using 4x or 10x objectives and short camera exposure times. As an alternative approach, a new flow layer device with &lt; 20 &#181;m height will be necessary to completely immobilize young larval stage C. elegans.
Tracking developmental phenomena in the same animal over a long timescale using high-resolution fluorescence imaging can result in increased autofluorescence. Every time-lapse imaging assay requires optimization of excitation intensity, exposure time, imaging time interval, animal recovery time, and food quality for physiologically relevant developmental information from C. elegans studies. We have selected &gt; 3 h time interval for developmental studies when using animals from L4 stages onwards. The device is capable of acquiring more frequent images (every 5-10 min for a few hours) or multiple frames per second (for &lt; 1 h) to study other dynamic processes such as organelle transport and distribution in C. elegans neurons3,23. Trapping and imaging of early developmental stages require more careful observation as repeated trapping with short time intervals without complete recovery can affect their health. During experiments, it was found that animals that required high pressure to move them into the flow layer show poor health and more autofluorescence over time. Animals with high body fluorescence, known to be associated with a high-stress level, were avoided for imaging studies. To maintain good physiology, animals were trapped in a straight position adjacent to the channel wall. Immobilizing the animals with their body across the channel width was avoided since animals become sick after their body is squeezed under 14 psi pressure in this position. To maintain a good signal-to-noise ratio during repeated imaging with oil objectives, the coverslip should be cleaned properly after every time point. Following special care during the fabrication process of the device and its application, the same devices could be reused over several imaging sessions.
This device could be useful for studies using mutants that could be sensitive to anesthetics. The approach presented can eliminate adverse anesthetic effects on growth and physiology to facilitate observation of morphological, functional, and behavioral defects in animals over a long period. The device is easy to fabricate and can be set up in any laboratory to address long-term developmental/cell biological questions in C. elegans that require intermittent high-resolution imaging.

Caenorhabditis elegans has proved to be a powerful model organism to study cell biology, aging, development biology, and neurobiology. Advantages such as its transparent body, short life cycle, easy maintenance, a defined number of cells, homology with several human genes, and well-studied genetics have led to C. elegans becoming a popular model both for fundamental biology discoveries and applied research1,2. Understanding cell&#39;s biological and developmental processes from repeated long-term observation of individual animals can prove to be beneficial. Conventionally, C. elegans is anesthetized on agar pads and imaged under the microscope. Adverse effects of anesthetics on the health of animals limit the use of anesthetized animals for long-term and repeated intermittent imaging of the same animal3,4. Recent advances in microfluidic technologies and their adaptation for anesthetic-free trapping of C. elegans with negligible health hazards enable high-resolution imaging of the same animal over a short and long period of time.
Microfluidic chips have been designed for C. elegans&#39;5 high throughput screening6,7,8, trapping and dispensing9, drug screening10,11, neuron stimulation with high-resolution imaging12, and high-resolution imaging of the animal12,13,14. Ultra-thin microfluidic sheets for immobilization on slides have also been developed15. Long-term studies of C. elegans have been performed using low-resolution images of animals growing in liquid culture to observe growth, calcium dynamics, drug effects on their behavior16,17,18,19, their longevity, and aging20. Long-term studies using high-resolution microscopy have been carried out to assess synaptic development21, neuronal regeneration22, and mitochondrial addition23. Long-term high-resolution imaging and tracing of cell fate and differentiation have been done in multichannel devices24,25. Several cellular and sub-cellular events occur over the time scales of several hours and require trapping the same individual at different time points during their development to characterize all intermediate steps in the process to understand cellular dynamics in vivo. To image biological process such as organogenesis, neuronal development, and cell migration, the animal needs to be immobilized in the same orientation at multiple time points. We have previously published a protocol for high-resolution imaging of C. elegans for over 36 h to determine where mitochondria are added along the touch receptor neurons (TRNs)23.
This paper provides a protocol for establishing a microfluidics-based methodology for repeated high-resolution imaging. This device, with a single flow channel, is best suited for repeated imaging of a single animal per device. To improve throughput and image many animals at once, multiple devices could be connected to the same pressure line but with separate three-way connectors controlling a single animal in each device. The design is useful for studies that demand high-resolution time-lapse images such as post-embryonic developmental processes, cell migration, organelle transport, gene expression studies, etc. The technology could be limiting for some applications such as lifespan and aging studies that require parallel growth and imaging of many late-stage animals. Polydimethylsiloxane (PDMS) elastomer was used for fabricating this device due to its biostability26, biocompatibility27,28, gas permeablility29,30, and tunable elastic modulus31. This two-layer device allows the growth of animals with continuous food supply in a microfluidic channel and the trapping of individual C. elegans via PDMS membrane compression using nitrogen gas. This device is an extension of the previously published device with the advantage of growing and imaging the same animal in the microchannel under a continuous food supply3. The additional isolation membrane network and a 2 mm wide trapping membrane enable efficient immobilization of developing animals. The device has been used to observe neuronal development, vulval development, and dendritic arborization in sensory PVD neurons. The animals grow without adverse health effects in the device and can be repeatedly immobilized to facilitate imaging sub-cellular events in the same animal during its development.
The entire protocol is divided into five parts. Part 1 describes device fabrication for the growth and imaging chip. Part 2 describes how to set up a pressure system for the PDMS membrane deflection to immobilize and isolate individual C. elegans. Part 3 describes how to synchronize C. elegans on a nematode growth medium (NGM) plate for device imaging. Part 4 describes how to load a single animal in the device and grow the animal inside the microfluidic device for several days. Part 5 describes how to immobilize an individual animal at multiple time points, capture high-resolution images using different objectives, and analyze the images using Fiji.

<table><tbody><tr><td>18 G needles</td><td>Sigma-Aldrich, Bangalore, India</td><td>Gauge 18</td><td></td></tr><tr><td>3-way stopcock</td><td>Cole-Parmer</td><td>WW-30600-02</td><td>Masterflex fitting with luer lock</td></tr><tr><td>CCD camera</td><td>Andor Technology</td><td>EMCCD C9100-13no</td><td></td></tr><tr><td>Circuit board film</td><td>Fine Line Imaging, Colorado, USA</td><td></td><td>The designs are printed with 65,024 dots per inch (DPI)</td></tr><tr><td>Convection Oven</td><td>Meta-Lab Scientific Industries, India</td><td>MSI-5</td><td></td></tr><tr><td>Coverslips</td><td>Blue stat microscopic cover glass</td><td></td><td>22mm x 10Gms</td></tr><tr><td>Ethanol</td><td>Hi media</td><td></td><td></td></tr><tr><td>Harris uni-core puncher 1mm</td><td>Qiagen</td><td>Z708801</td><td></td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma-Aldrich, Bangalore, India</td><td>440191</td><td></td></tr><tr><td>Hot plate</td><td>&amp;nbsp;IKA</td><td>RCT B S 22</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>26895</td><td></td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Laser Scanning Microscope</td><td>ZEISS</td><td>LSM 5 LIVE</td><td></td></tr><tr><td>Micropipette tips</td><td>Tarsons</td><td></td><td>0.5-10 &amp;micro;L micropipette tips are used for food supply</td></tr><tr><td>Negative Photoresist-1</td><td>Microchem</td><td>SU8-2025</td><td>http://www.microchem.com/Prod-SU82000.htm</td></tr><tr><td>Negative Photoresist-2</td><td>Microchem</td><td>SU8-2050</td><td>http://www.microchem.com/Prod-SU82000.htm</td></tr><tr><td>Nitrogen gas</td><td>Local Supplier</td><td>Commercial nitrogen gas</td><td>Cylinder volume of 7 cubic meter</td></tr><tr><td>PDMS (Curing solution)</td><td>Dow Corning Corporation, MI, USA</td><td>&amp;nbsp;Sylgard curing solution</td><td>curing agent</td></tr><tr><td>Petri plates</td><td>Praveen Scientific Corporation</td><td></td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma, NY, USA</td><td>&amp;nbsp;PDC-32G</td><td></td></tr><tr><td>Razor and blades</td><td>Lister surgical Blade</td><td></td><td></td></tr><tr><td>Silicon Elastomer (Base)</td><td>Dow Corning Corporation, MI, USA</td><td>Sylgard 184 base</td><td>elastomer base</td></tr><tr><td>Silicon tubes</td><td>Fisher Scientific</td><td></td><td>Plastic tubes with the inner diameter 1.59 mm and the outer diameter 3.18 mm</td></tr><tr><td>Silicon wafer</td><td>University Wafer, MA, USA</td><td>[100] orientation, 4-inch diameter</td><td>Small pieces (2 mm &amp;times; 2 mm) were cut from 100 mm diameter wafer</td></tr><tr><td>Spin Coater</td><td>SPS-Europe B.V., The Netherlands</td><td>SPIN 150</td><td></td></tr><tr><td>Spinning Disk microscope</td><td>Perkin Elmer ultra-view VOX system</td><td>CSU-X1-A3 N</td><td>The system was equipped with four (405/488/561/640 nm) lasers and controlled with the Volocity software package.</td></tr><tr><td>SU8 developer</td><td>Microchem, MA, USA</td><td>SU8 Developer</td><td></td></tr><tr><td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td><td>Sigma-Aldrich, Bangalore, India</td><td>448931</td><td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane vapor is toxic</td></tr><tr><td>UV lamp</td><td>Oriel Instruments, Bangalore, India</td><td></td><td>200 Watt and collimated UV light source</td></tr><tr><td>Volocity software</td><td>Perkin-Elmer</td><td></td><td>Image analysis</td></tr></tbody></table>

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.

Device characterization: The growth and imaging device consists of two PDMS layers bonded together (Figure 1) using irreversible plasma bonding. The flow layer (pattern 1) which is 10 mm in length and 40 &#181;m or 80 &#181;m in height allows us to grow the animal in liquid culture (Figure 1A). The trapping layer (pattern 2) has a 2 mm wide membrane (Figure 1B) for immobilizing the animal for high-resolution imaging. The mask for the trapping layer also creates a pair of isolation membranes for restricting animal movement within a section of the flow channel between successive imaging time points. The trapping membrane of the device is adapted from our previous imaging device3. The current device (Figure 1C,L) includes the addition of an isolation membrane and an increase in the width of the trapping membrane to 2 mm for the efficient immobilization of the same animal over multiple time points.
To test the device, clean distilled water was filled in the microfluidic channels connecting the trapping membranes (Figure 1D,E, and Supplementary Figure 3) and pressurized using 14 psi nitrogen gas, also used for trapping an animal under a 2 mm thick PDMS membrane (Figure 1H,K,L). A single animal was picked from an NGM plate using a micropipette and loaded into the flow channel (Figure&#160;1F). The growth channel was filled with bacteria resuspended in S media (Figure 1F,G,I,J). A constant flow was maintained throughout the duration of imaging by adjusting the media heights in the pipette tips connected to the device inlet and outlet while monitoring the animal in the flow channel between the two isolation membranes. Freshly prepared OP50 solution was filled in the micropipette tips daily to ensure a healthy food source for the animal inside the microchannel. Fresh food source and gas permeable PDMS material ensure sufficient oxygen supply inside the microchannel30,29. The growth of animals in the device was tracked by using either cell-specific markers or markers that show cell lineage or variable cellular expression patterns during development. The animals were immobilized under the trapping membrane using 14 psi nitrogen gas and holding worms in a straight position along the channel wall. The animal grew slower inside the microfluidic channel compared to on an NGM plate23.
Cell lineage study using the long-term imaging device: To assess C. elegans development inside the microfluidic device, we grew the PS3239 (ZMP-1::GFP) strain32 with constant food supply to track vulva development at different time points of their growth. ZMP-1 codes for a zinc metalloprotease and expresses in the anchor cell in the L3 stage, in vulD and vulE cells in the L4 stage, and in vulA in day 1 (1D) adult animals (Figure 2A). The changes in expression pattern represents an example of temporal gene regulation where the same gene is expressed in different cells at different stages of development. To observe vulval development, the animal was first immobilized and the vulval region was then imaged across multiple z planes every 8-10 h from L3 onwards until adult stages. ZMP-1::GFP is expressed in different vulval&#160;cells according to the developmental stage (Figure 2B). The high-resolution fluorescence images of the vulval cells from the animals growing inside the microfluidic device demonstrate normal vulval development and ZMP-1::GFP expression and localization are similar to prior&#160;reports32.
Tracking neuron development using long-term growth and imaging device: To demonstrate the use of the device for sub-cellular imaging from an individual animal, the development of two mechanosensory neurons PVD and TRNs was monitored. The NC1686 strain expressing wdIs51 that expresses GFP in the two PVD neurons was used34,36. Each PVD neuron shows menorah-like branched dendritic architecture comprising of primary (PP), secondary (SP), tertiary (TP), and quaternary (QP) processes at L2 (14-16 h), late L2 (20-22 h), L3 (24-26 h), and L4 (36-42 h) developmental stages, respectively (Figure 3). The PVD cell body is present posterior to the vulva. It sends out one axon and two primary dendritic processes that give rise to SP and TP, which in turn give rise to QP dendritic branches that innervate the body wall muscles. Late L3 and L4 stages show high arborization37,34. We grew single NC1686 animals inside the microfluidic device and fed them continuously with bacterial food. The animals were immobilized during L2 up to 1D stage of development and the PVD neurons were repeatedly imaged every 8-12 h using 60x, 1.3 NA oil objective to count the number of SP, TP, and QP branches in three dimensions. The extent of branching increased during development as shown in Figure 3B. The numbers of SP and QP at different stages of the worm development increased with age (Figure 4A) as has been seen in prior studies37. L2 animals showed 10 &#177; 3.8 (n = 9) SPs but no QP, when measured using the device (Figure 4A). We placed&#160;C. elegans in a drop of 3 mM levamisole, an anesthetic that causes contraction of body wall muscle and paralysis, to minimize adverse effects on organelle transport while reducing animal movements and causing sufficient paralysis, required for high-resolution imaging3,4. The SP values (4 &#177; 1.6, n = 25, p = 0.15) were not significantly different when measured from similar staged animals grown on NGM plates and imaged using 3 mM levamisole on an agar slide (Figure 4B). The data suggest that the device immobilization enables high-resolution imaging of complex neuronal architectures and does not affect their development. L3 stage animals have a small number of QP that increases in number with development. 1D adults showed 67 &#177; 2.8 QPs (n = 5) in animals grown in the device. Previous studies have shown the formation as well as a retraction of PVD processes during development known as self-avoidance, where they reduce their branch numbers38. The reduction in SP at 51 h (1D) after hatching could be the result of such retractions. Animals growing on NGM plates and imaged using 3 mM levamisole showed a similar branching number trend for comparable stages of development (Figure 4A,B). Further, the distance between the two PVC cell bodies, one present in the tail and the second present near the vulva, increases as they move apart in animals grown in the device or those grown on NGM plates (Figure 4C,D). Throughout the imaging process, the animals remained healthy and could lay eggs even after the animals were immobilized under the membrane repeatedly over this long period.
Using this device, we were able to immobilize the animal in the same orientation and image the identical neuron and its architecture with high resolution even with faint GFP expression. To demonstrate the utility of our growth and immobilization technology, the development of the TRNs from L3 to adult was imaged using jsIs609 (mec-7p::MLS::GFP) animals. The posterior TRNs are present at the lateral side of the body and are named PLMR and PLML which corresponds to the right and left sides of the animal. We imaged the same animals growing in the microfluidic chip and immobilized them at 12-14 h intervals. The montage represents the entire PLMR neuronal process at successive time points highlighting both mitochondria and the neuronal process (Figure 5A). The total neuronal process length was calculated and found to increase with a slope of 10.4 &#181;m per h (Figure 5B). This rate of increase in the neuronal process length&#160;agrees with a previous report of ~10 &#181;m/h18,31,39,40,41. We also observed the addition of new mitochondria in the growing process and the change in the synaptic branch point position with respect to the cell body as reported earlier23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63136/63136fig01.jpg" /> 
Figure 1: A simple microfluidic chip for long-term growth and imaging of C. elegans. (A) The flow layer consists of a 10 mm long microfluidic channel (pattern 1) in a thin PDMS layer. (B) The trapping layer consists of a 2 mm thick immobilization channel and a pair of thin isolation channels in the bulk PDMS layer. (C) The two PDMS layers are bonded together along with a thin cover glass to make the device. (D, E) The trapping and isolation control channels are filled with deionized (DI) water, under compressed nitrogen (N2) gas. (F) Age synchronized C. elegans are picked from NGM plates and loaded inside the flow layer of the device. (G) Food is provided using two micropipette tips at the inlet and outlet reservoirs. (H) The animals are free to move within the two isolation channels and are trapped under the immobilization membrane. (I) Image of the growth and imaging device with food supply through two micropipette tips. (J, K) Images of a freely moving and immobilized C. elegans inside the microfluidic channel. Scale bar is 1 mm (I) and 200 &#181;m (J, K). (L) Schematic of the device cross-section showing the heights of the flow channel (FC), PDMS membrane (M), and control channel (CC). <a href="https://www.jove.com/files/ftp_upload/63136/63136fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63136/63136fig02.jpg" /> 
Figure 2: Tracking the expression of a vulval marker in C. elegans developing inside the device. (A) Schematic of vulval cells expressing ZMP-1::GFP in PS3239 animals during development. The GFP signal appears in the anchor cell at the L3 stage, in the vulD and vulE cells at the L4 stage, and in vulA cells at the adult 1D stage. (B) Images of PS3239 animal growing inside the microfluidic chip and immobilized every 8-10 h to capture fluorescence images of ZMP-1::GFP expression during vulval development from L3, L4, and 1 day (1D) adult. Abbreviations: AC = anchor cell, A = vulA, D = vulD, E = vulE. Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63136/63136fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63136/63136fig03.jpg" /> 
Figure 3: Imaging of individual wdIs51 C. elegans to track PVD neuronal development. (A) Schematic of PVD neuron growth and dendritic arborization from L2 to L4 stage. Green circle shows PVD cell body (CB, yellow arrow). The dendrites show the emergence of primary processes (PP) at L2 from both the anterior and posterior side of the CB, secondary processes (SP) during late L2, tertiary processes (TP) in L3, and quaternary processes (QP) during L4 stages. (B) Images of PVD neurons from animals growing inside a microfluidic device. (C) Images of PVD neurons from animals grown on an NGM plate and immobilized with 3 mM levamisole (Lev). Scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63136/63136fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63136/63136fig04.jpg" /> 
Figure 4: Comparison of PVD development in device grown animals and animals grown on NGM plates. (A) The average number of secondary processes (SP) and quaternary processes (QP) from the same animals growing inside the microfluidic device. The values are calculated at 16 h (L2), 24 h (L3), 36 h (early L4), 42 h (late L4), and 51 h (1D adult). (B) The average number of SP and QP from a different batch of animals growing on NGM plates and anesthetized with 3 mM levamisole. (C) Average separation between the two PVD neuronal cell bodies from the same animal growing in the microfluidic device. (D) Average separation from different sets of animals growing on NGM plates and anesthetized with 3 mM levamisole. Data represented as mean &#177; SEM (n &#8805; 10 for 3 mM levamisole and n &#8805; 6 for device immobilized animals). The statistical significances are calculated by one-way ANOVA and denoted as p-value &#60; 0.05 (*), p-value &#60; 0.005 (**), and p-value &#62; 0.05 (ns). <a href="https://www.jove.com/files/ftp_upload/63136/63136fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63136/63136fig05.jpg" /> 
Figure 5: High-resolution imaging of touch receptor neurons (TRNs) from animals growing inside the microfluidic device. (A) Montage of neuronal processes from a single animal imaged at different times. The cell body (CB, arrow), the synaptic branch point (BP, arrowhead), and the neuron tip (Tip, up arrow) are labeled. The neuronal process and position of each mitochondrion are manually traced using Fiji. Scale bar is 10 &#181;m. (B) Average neuronal process length at different imaging time points. Data represented as mean &#177; SEM (n = 8). The statistical significance is calculated using one-way ANOVA and denoted as p-value &#60; 0.005 (**) and p-value &#62; 0.05 (ns). A regression equation is fit with a slope of 10.4 &#181;m neuronal process elongation per hour, R2=0.9425. <a href="https://www.jove.com/files/ftp_upload/63136/63136fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Screenshots of microscope settings for fluorescence imaging of C. elegans. (A) A panel of the image analysis software shows the highlighted sections for setting up the scan speed, filter set, and objective for imaging. (B) The software is then used to choose the 488 nm laser with 7% of the full laser power. (C) The image analysis software can take a single image frame or a series of time-lapse images and store them for future analysis. <a href="https://www.jove.com/files/ftp_upload/63136/63136JoVE Revision2 Supp Figure1.tif" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Screenshots of FIJI software showing the analysis steps for counting PVD branches in wdIs51 animals. (A) Shows PVD image opened in Fiji ImageJ software and link to the cell counter plugin. (B) Cell counter window to initialize the counter for an image. (C) Selection of counters to mark branches that are included and to avoid multiple counting. (D) Results window showing the total number of branches under each category. <a href="https://www.jove.com/files/ftp_upload/63136/63136JoVE Revision2 Supp Figure2.tif" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Schematic of the growth and imaging device. Two micropipette tips, filled with different volumes of bacteria food solution (yellow), are inserted in the inlet and outlet punches of the flow channel at the bottom layer. The difference in the heights of food solutions in the tips causes the solution to flow inside the channel which in turn supplies bacteria to the animal for its growth. The trapping and isolation channels (blue) are filled with distilled water and compressed using a nitrogen gas (set at 14 psi) supply. The isolation channel is always connected to 14 psi. The pressure on the trapping membrane is switched between 0 (animal is free to move and feed) and 14 psi (animal is immobilized for high-resolution imaging) using a three-way connector. <a href="https://www.jove.com/files/ftp_upload/63136/63136JoVE Revision Supplemenatry Fig 3 (1).tif" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans at JoVE.com

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.

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 ...

a-simple-microfluidic-chip-for-long-term-growth-imaging

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.9K Views.  TIFR. 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. 

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

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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 ...

Biology

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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

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Chapters in this video

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices