Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging

Our lab is interested in the mechanisms regulating neutrophil migration and activation in tissue injury, infection, and cancer with a focus on characteristics and the biological significance of intracellular calcium signaling in wound healing.

[Shelly] Conventional microfluidic design methods are costly and complex, so slowing progress in fields like developmental biology that require diverse environments. We saw a more rapid and efficient approach for developing and testing individual designs. So this construction method provides greater design flexibility and spatial resolution than similarly simple protocols while enabling faster and easier iteration than standard PDMS devices do. Using it, we developed the RADISH to simultaneously image multiple zebrafish embryos within a single field of view.

With this entrapment device that supports high-resolution life imaging, we're set to understand how diverse signals including chemical and the mechanical stimuli, integrated to coordinate inflammation and wound healing.

[Instructor] To begin, design the pattern for the device using the software of choice. Depending on the age and size of the embryo, adjust the width of the channel and the size of the central imaging chamber for the best fit. Save each layer as an individual image file with identical dimensions. Now, initialize the cutting machine according to the manufacturer's instructions. In the design workspace, select the newly uploaded designs. Group patterns intended for the same thickness of PET sheet together in the same cut. Confirm the number and type of patterns in the cut, then select the cut material. Next, attach the PET sheet to the cutting mat at the desired thickness pressing firmly to remove air bubbles, and load the mat into the cutting machine. Follow on-screen instructions to prompt the cutting machine to begin the cut. Then, remove the finished cuts from the cutting mat. Now, align the layers on a cover slip glass, either unassisted or with pre-cut registration marks. Secure the layers with office tape to avoid shifting during lamination. Depending on the device size and laminator capacity, mount small designs to a larger backing with more tape to avoid jamming or clogging the laminator feed. Afterward, turn on the laminator and laminate according to the manufacturer's instructions. Remove the office tape before aligning the next layer. Using cyanoacrylate glue, secure the device to a 35-millimeter dish drilled with a three-quarter inch hole. Then waterproof the laminate by thoroughly coating the outer device edges with cyanoacrylate glue. Use enough glue to fully cover the outer edges without gaps. To begin, raise zebrafish embryos to at least two days post fertilization in E3 or another suitable embryo rearing medium. Then, anesthetize the embryos using 164 milligrams per liter of MS-222. Next, fill the holding dish containing the mounted device with five milliliters of E3 or another embryo medium containing 164 milligrams per liter of MS-222. Using a transfer pipette, deposit one anesthetized embryo into each holding chamber of the device. Then, using a hair loop, orient the embryos with the tail of each embryo protruding into the central imaging chamber, and the yolk immobilized in the wedge-shaped channel. If desired, secure the head of the embryo by adding 1.5% low melt agarose into the holding chamber using a micro pipette. Finally, move the device with embryos to the imaging system of choice to commence imaging, Select the appropriate imaging system and objective for the experiment. Adjust the sample focus and imaging parameters for optimal signal to noise ratio and acquisition speed. To wound the embryo via manual transection, apply pressure with a scalpel blade across the tail fin tissue at the tip of the notochord under a stereo microscope. Then, return the device to the stage. To wound the embryo via laser stimulation, define a region of interest near the edge of the tail fin fold, and stimulate according to the available laser power. Image the embryo at room temperature using the GFP and RFP channel with a frame interval of one minute for a total of one hour. If necessary, release the embryos after imaging by gently suctioning near the head using a transfer pipette or by swirling the dish.