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

The overall goal of this imaging device fabrication is to create a modifiable platform for microscopic analysis of wound healing in live zebrafish larvae. This method can help answer key questions in the wound healing field such as what is the contribution of collagen fiber reorganization and cellular responses to subsequent tissue regrowth after injury. The main advantage of this technique is that wounding and imaging occurs within a single device, while tail regrowth is not impeded by the device.

Additionally, the multiple functional compartments of the zWEDGI could be modified independently to accommodate different larval stages and an array of experimental protocols. To begin, model the PDMS component of the device with the desired geometries and attributes according to the text protocol. After printing the molds using a photopolymer 3-D printer, clean the molds using a fine brush, denatured alcohol in a spray bottle, and compressed air.

Gently scrub and remove the uncured resin, particularly any material from the microchannel regions. Then, postcure the molds in a UV postcure apparatus for 60 minutes on each side, as uncured resin is toxic to zebrafish larvae. Using 200 grit sandpaper, sand the cavity side of the mold on a flat surface until all the ceiling surfaces are in contact with the sandpaper.

With 400 and 600 grit sandpaper, lightly sand progressively to produce smooth, flush surfaces across all geometry facings of the mold. Then, use a dial indicator to measure the depth of the cavity after sanding to verify that it is close to the design depth. Clean the molds and glass cover disks by placing them in an ultrasonic cleaner filled with water for 30 minutes or by flushing them under running water.

Use isopropyl alcohol and filtered compressed air to clean and blow dry the molds and covers. Prepare a clean bench as a place to fabricate the devices to minimize contamination from airborne debris. Make the PDMS by pouring base and activator at a ratio of 5:1 into a plastic cup.

With a wooden craft stick mix well for two minutes, stirring the gel over onto itself like kneading bread. Degas the mixture in a vacuum desiccator for 25 to 45 minutes until all bubbles are gone. Then, using a 10 milliliter syringe, fill the cavity of each 3-D printed mold with approximately 0.75 milliliters of PDMS until the mold slightly overflows with a meniscus.

Degas the filled molds for 45 minutes to remove additional bubbles that may have formed when filling. Apply a glass cover disk on top of the PDMS filled mold, pressing the disk down at an angle to prevent bubbles from being trapped. Allow excess PDMS to be expelled as the glass disk cover is applied.

Then, use a small ratchet clamp to hold the cover disk tightly to the mold. For efficiency, a multi-clamp device can be used. Cure the PDMS device and the clamped molds at 100 degrees Celsius in an oven for at least 90 minutes.

Then, remove the molds from the oven, and allow them to cool until they can be easily handled. Clamp the mold containing the cured PDMS device in a bench vice so that the mold's geometries are facing up, parallel to the working station bench. Start to remove the PDMS device by using flat tipped tweezers to release the PDMS pull tap from the mold.

Then, work around the perimeter of the mold with the tweezers. The PDMS must be cured for at least 90 minutes at 100 degrees Celsius or else it will remain tacky and stick to the mold. A good indication that you've cured the device long enough is that you'll actually see an air bubble at the interface between the mold and the PDMS device, indicating that it will be easily removed.

Use filtered, compressed air and tweezers to gently lift the device out of the mold by holding on to the pull tab and blowing air under the device. Next, place the PDMS device upside down onto the inside of the cover of a glass bottom dish so that the restraining tunnel wedges are touching the plastic. Then, place the dish cover with upside down zWEDGI and the corresponding glass bottom dish into a plasma cleaner with the inner glass facing upward.

Evacuate the plasma cleaning chamber until the pressure reaches 500 millitorr. Set the radio frequency, or RF power, to high and expose the device and the dish to RF frequency for approximately two minutes. Then, slowly depressurize the chamber and return the device and the dish to a clean room hood.

Remove the PDMS device from the dish cover. Then, flip over the PDMS zWEDGI to the correct orientation on the glass by positioning it carefully onto the center well of the glass bottom dish. To ensure PDMS adherence to the glass, use the back end of the tweezers to apply pressure around the minute geometries of the microchannels, smoothing the PDMS out toward the edges.

Lightly press down on the PDMS device to ensure air bubbles are not trapped beneath the device. Add 70%ethanol to the dish and using a micropipette, rinse the channels including through the restraining tunnel. Then, remove the ethanol and use double distilled water to rinse the device two or three times before allowing it to air dry.

Fill the channels with skim milk, and incubate the device for 10 minutes at room temperature to minimize adherence of the larvae to the glass cover slip of the dish. Then, gently submerge the device several times in double distilled water to rinse, and allow it air dry upside down. After anesthetizing larvae according to the text protocol, use a few microliters of tricaine E3 to pre-wet the channels of the device.

Using a wide orifice pipette tip, pick up a single larva and deposit it into the loading channel. Then, with a pipette tip or similar tool, orient the larva in the loading chamber with the dorsal side facing the straighter edge of the loading chamber, and the tail facing the restraining tunnel. Carefully withdraw fluid from the wounding chamber, allowing the larva to flow into the restraining tunnel.

Remove most of the liquid while maintaining moisture around the larva. The larva must be properly oriented into the restraining tunnel as most of the liquid is removed. But sufficient liquid must remain around the larvae.

Orientation can be adjusted after agarose addition but must be accomplished before the agarose solidifies. Pipette 1%LMP agarose in tricaine E3 over the larva's head, filling the loading chamber, and allow agarose to solidify with the larva in the proper position. Add tricaine E3 to the wounding chamber as needed to maintain hydration.

Then, repeat this loading process for the remaining two channels. Using a syringe needle, carefully remove any agarose that seeped through the restraining tunnel into the wounding chamber. For wounding, short term imaging, or wound treatment isolation, add more tricaine E3 over the agarose.

To wound the larvae in the wounding chamber, use a steril scalpel blade to transect the tail fin posterior to the notochord. Add additional tricaine E3 if needed and replace the culture dish lid. Install the zWEDGI device with anesthetized larvae onto an inverted microscope in a stage insert that will accommodate the 60 millimeter glass bottom dish.

Locate the tail of the larvae in the uppermost channel, rotating the dish as needed to get the tail in the desired position. Image the larva as required for the specific experiment. At the conclusion of the experiment, remove the zWEDGI dish from the microscope.

After removal of the larvae and agar according to the text protocol, use ethanol and distilled water to clean the zWEDGI as demonstrated earlier, and set it upside down to air dry. Store the covered device in a cool, dry location. Shown here are images using multiphoton microscopy for second harmonic, or SHG imaging of collagen fibers in the caudal fin to illustrate the imaging capabilities of the zWEDGI.

Prior to wounding, the SHG detected collagen fibers that radiate outward from the notochord to the fin tip. Shortly after wounding, the distance between the tip of the contracted fin and the center of the fin increases with wound relaxation. The zWEDGI permits the collection of three dimensional data over time, providing a more complete view of the dynamic changes occurring in the structure of the extracellular matrix.

The 3-D nature of the data collection permits spatial reconstruction using rendering software. These reconstructions and the subsequent rotation options illustrate that the contraction and relaxation of the fin occurs not only in the XY plane of the image collection, but also in the Z axis, as the tail flattens from the upward curl of the contracted state. Once the reusable molds have been printed, the fabrication technique can be done in three to four hours.

While attempting this procedure it's important to remember to ensure no air bubbles are trapped in the PDMS before curing and to cure the devices long enough before trying to remove them from the molds. Following this procedure, other methods such as localized drug application, antibody labeling, or RNA and protein purification can be performed in order to answer additional questions like how the presence of drugs or inhibitors impact wound healing. After its development, this technique paved the way for researchers in the field of wound healing to explore collagen dynamics, immune cell response, and tissue regrowth in the zebrafish larvae.

After watching this video you should have a good understanding of how to fabricate a zWEDGI and use it for wounding and live imaging of zebrafish larvae. Don't forget that photopolymer 3-D printing resins are hazardous to humans and toxic to zebrafish. Precautions must be taken such as wearing gloves and using a face mask while handling uncured 3-D printed parts, and ensuring that all resin remaining on the prints is cured before attempting to fill the molds with PDMS.