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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Research

JoVE Journal

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

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 ...

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&#039; 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 ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

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 ...