Name: a simple microfluidic chip for long-term growth and imaging of caenorhabditis elegans
Uploaded: 2022-04-11T13:00:00
Description: Watch this Scientific Journal Video about A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans at JoVE.com

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.

1. Fabrication of growth and imaging device
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	<li>SU8 mold fabrication
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		<li>Design patterns 1 (flow layer) and 2 (control layer) using rectangular shapes in a word processing software (or a computer-aided design CAD software) and print the photomasks with the help of a laser plotter with a minimum feature size of 8 &#181;m on polyester-based film (Figure 1).</li>
		<li>Cut silicon wafers in 2.5 cm &#215; 2.5 cm pieces and clean them with 20% KOH for 1 min. Rinse...

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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 durin...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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 &#181;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>&#160;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>&#160;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 &#215; 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...

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 ...

The main advantage of this technique is that the animal can grow freely inside the device and can be trapped for high resolution imaging. The device can be reused several times. While making the device, cleanliness is the most important factor to be able to fabricate dust-free devices to avoid leakage during the device operations.To begin design pattern one for the flow layer and pattern two for the control layer using rectangular shapes in a word processing software and print the photo masks with the help of a laser plotter with a minimum feature size of eight micrometers on polyester based film. Cut silicon wafers into square pieces of 2.5 centimeters in height and width. Clean them with 20%potassium hydroxide for one minute and rinse the wafers in deionized water.Use one wafer for the flow and the other for the control layer. Dry the pieces with 14 P-S-I compressed nitrogen gas followed by dehydration on a hot plate at 120 degrees Celsius for four hours. After cooling take one silicon piece, put it on the chuck of a spin coder and turn on the vacuum to hold the wafer in place.Put approximately 20 microliters of hexamethyldisilane on the silicon piece and coat it using the spin coder at 500 R-P-M for five seconds followed by 3000 R-P-M for 30 seconds. To get a uniform photoresist thickness of approximately 40 micrometers specific to the flow layer, coat the silicon wafer with approximately 1.5 milliliters of negative photoresist one using a spin coder at 500 R-P-M for five seconds followed by 2000 R-P-M for 30 seconds. Repeat the coating of the silicon piece with hexamethyldisilane and negative photoresist one for the second wafer to obtain a uniform photoresist thickness of approximately 40 micrometers specific to the control layer.Alternatively, to increase the thickness of the flow layer to approximately 80 micrometers for older animals coat silicon wafers with approximately 1.5 milliliters of negative photoresist two, using the spin coder at 500 R-P-M for five seconds followed by 2000 R-P-M for 30 seconds. Bake both the previously photoresist coated silicone pieces on a hot plate at 65 degrees Celsius for one minute followed by 95 degrees Celsius for 10 minutes. After cooling, put soft baked silicone pieces on the exposure stage of the U-V illuminator with the photoresist coated surface facing the U-V lamp.Expose the two pieces separately to U-V for 15 seconds using a 200 watt lamp through a photo mask with patterns one and two to get flow and control layers respectively. Bake the two exposed silicone pieces as previously described with coated layers facing up. After cooling pieces, develop the patterns by soaking the silicone pieces in the photoresist developer solution for 20 minutes.Once the pattern is visible, rinse the pieces with pure isopropyl alcohol and gently blow dry using nitrogen gas. Keep the silicone pieces in a desiccator with the coated surface facing up. Expose the pieces to silane vapors by pouring 50 microliters of pure T-C-P-F-O-S on a glass slide.Place the slide inside a desiccator and incubate for two hours. Make P-D-M-S in a plastic cup by mixing the elastomer base with the curing agent and constantly stir for three minutes. Degas the P-D-M-S mix in a desiccator for 30 minutes to remove all air bubbles.Place the control layer silicon wafers in a Petri dish and pour a five millimeter thick P-D-M-S mix layer on the silicon pieces. After P-D-M-S pouring process, repeat degassing the P-D-M-S mix. Place the silicon wafer with flow layer facing the spinner truck applying 200 to 500 millitorr vacuum pressure.Pour approximately one milliliter of P-D-M-S on the silicon wafer. Encode it using a spin coater to get an approximately 80 micrometer thick layer. Bake the two silicon wafers with the spin coated P-D-M-S and poured P-D-M-S layers at 50 degrees Celsius in a hot air convection oven for six hours.After cooling down the pieces cut the five millimeter thick P-D-M-S layer from the silicon piece around the control layer using a sharp blade and peel it off the silicon substrate. Punch two holes of approximately one millimeter diameter using a Harris puncher at the reservoir of the P-D-M-S block to connect the immobilization channel and isolation channel inlets to the gas lines for P-D-M-S membrane deflections. Place the silicon piece with the spin coated P-D-M-S layer on pattern one with the P-D-M-S coated surface facing up on a plastic tray.Keep the punched P-D-M-S block with pattern two on the tray with molded side facing up. Keep the plastic tray inside a plasma cleaner and expose the two P-D-M-S surfaces to 18 watts air plasma for two minutes by applying low vacuum. Take out the two plasma treated blocks and gently bind the blocks by pressing the plasma treated surfaces of patterns one and two together.Bake the bonded patterns at 50 degrees Celsius for two hours in a hot air convection oven. After taking the bonded device out of the oven, cut it out of silicon wafer with pattern one and pattern two and punch holes in the inlet and outlet reservoirs of the flow layer using the Harris puncher. Place the bonded P-D-M-S block with the flow layer facing up on a plastic tray and keep a clean cover glass on the same tray.Expose the blocks in the cover glass to 18 watt air plasma for two minutes. Adjust vacuum pressure to see a violet chamber. Place the plasma exposed P-D-M-S block on the cover glass and bake the bonded structure in an oven at 50 degree Celsius for two hours.Store the device in a clean chamber for any future experiment. Take the device, put it on a stereo microscope and attach the tubings. Connect a microflex tube to a compressed nitrogen gas line and a three-way connector on the other end.Connect tubes one and two of the three-way connectors to the trap in isolating membranes respectively. Connect two microflex tubes to the two outlet ports of the three-way stopcock. Connect the other end of the two tubes to an eight millimeter long 18 gauge needle.Fill the flow layer with M-9 buffer using a micro pipette through the inlet port. Fill both tubes with deionized water through the end connected to the needle. Insert the two needles into the punched holes connecting the isolating and trapping membrane.Open the nitrogen gas regulator at 14 P-S-I and turn the three-way valve from tube one to push the water into the device through the microfluidic channels in the control layers, namely the trap and isolation membranes. Release the pressure using the three-way stopcock, once the channels are filled with water and primed. Remove the bubbles by flowing additional media through the flow channel.The figure shows images of P-S-3-2-3-9, animal growing inside the microfluidic chip and immobilized every eight to 10 hours to capture fluorescence images. Variation in expression pattern represents an example of temporal gene regulation where the same gene is expressed in different cells at different stages of development. Imaging of individual W-D-I-S-5-1 genotype of caenorhabditis elegans shows menorah-like branched dendritic architecture in each P-V-D neuron comprising primary, secondary tertiary, and quaternary processes at L-2, Late L-2, L-3 and L-4 developmental stages respectively.The figure herein compares P-V-D development and device grown animals, and those grown on N-G-M plates. The numbers of S-P and Q-P at different stages of the worm development increased with age. Caenorhabditis elegans was treated with a drop of three millimolar levamisole to cause sufficient paralysis required for high resolution imaging.The S-P values were not significantly different when measured from similar staged animals grown on N-G-M plates and imaged using three millimolar levamisole on an Agar slide. Further, the distance between the two P-V-C cell bodies, one present in the tail, and the second present near the vulva, increases as they move apart in both animals grown in the device and those grown on N-G-M plates. The figure represents the high resolution image of touch receptor neurons from animals growing inside the microfluidic device.The montage represents the entire P-L-M-R neuronal process at successive time points highlighting mitochondria and the neuronal process. The graph represents total neuronal process length which was observed to increase with a slope of 10.4 micrometers per hour. The micro-fabricated device uses a thin P-D-M-S membrane deflected in presence of high pressure nitrogen gas to immobilize and hold C-elegans in place for high resolution imaging.This procedure can enable longitudinal studies of cellular and subcellular processes in C-elegans that need intermittent observations over a long period of time.

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