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.
