The overall goal of this microfluidic spaced culturing method, is to grow plants directly from seed in a structured environment that can also be used for controlled treatment and high resolution imaging of the seedlings'roots. This method can help answer key questions in the plant biology field such as, how plant root growth responds to physical and chemical cues at multiple length scales. The main advantage of this platform is that the repairs are confined to a single plain, making it impossible to treat and image them without losing optical focus.
Assisting me today will be Dr.Sahar Hasim, she's a research associate professor at the University of Tennessee. To begin, pour a 10:1 ratio of PDMS to curing agent onto a pre-fabricated and selenised master wafer. Degas the PDMS in a vacuum chamber and then, cure the polymer in an oven at 70 degrees celsius for one hour.
Next, use a scalpel to cut the PDMS devices and peel them from the master. Then, use a 1.5 milliliter biopsy to punch the seed inlet at a 45 degree angle, to encourage root growth into the main channel. Also, use the same punch to clear the treatment inlets.
Use adhesive tape to remove any debris from the device and place it design side down on a glass cover slide. Once assembled, autoclave the devices. Place the Arabidopsis thaliana seeds in a microfuge tube and add a mixture of bleach and detergent to sterilize the seeds.
Then, wash the seeds four times with sterile water. Next, stratify the seeds by refrigerating them in the microfuge tube overnight or store them for up to one week at four degrees celsius. Prior to using the sterile devices, place them into a vacuum degassing chamber, to remove air from the gas permeable PDMS and fluidic channels.
This will improve the subsequent filling of the devices. Remove the devices from the vacuum chamber and immediately submerge them in a petri dish filled with liquid one quarter strength plant based media, at a pH of 5.7. Using a pipette, pull liquid through the inlets to fill the device.
Make sure no air bubbles remain within channels by visually inspecting them before transferring them to individual fresh, dry petri dishes. Next, pour hot agar around the device until the agar level is almost flush with the top of the device. In a sterile environment, use a small pipette and transfer one sterilized seed to the inlet of each device.
Then, cover each petri dish with wax film and place them in a light dark cycling growth chamber at room temperature. Orient the petri dish in the growth chamber at room temperature, vertically, so gravity will encourage the roots to grow through the channel. At the desired time and the seedling's development, use a pipette to add a prescribed amount of the experimental treatment to each of the eight side ports on the devices.
When finished, reseal the petri dishes with wax film. Then, return them to the growth chamber. Adjust the magnification of an inverted bright field microscope, at between 4x and 20x magnification.
Place the entire petri dish containing the device and seedling under the microscope for bright field or differential interference contrast imaging. Optimize the lighting conditions by adjusting exposure times, lamp brightness, aperture and the polarity of the light to elucidate the morphological features that are of interest. Then, drive the stage to the desired area of the root and focus on the root or root hair of interest.
Acquire a single time point or a time series of images to visualize the growth of root hairs. For the time series, image the growing root hairs once per minute. To visualize the growth of the main root, acquire one image every 30 minutes.
When finished, return the petri dish and the seedlings to growth chamber, in a vertical position. Once the seedling has grown for the desired time, use a pair of forceps to remove the cover slip and the device from the agar. Then, wet a laboratory tissue with ethanol and use it to clean off the bottom of the cover slip.
Apply the proper oil to a 63x oil immersion objective and place the cover slip down on the microscope stage. Then, raise the stage to contact the immersion media on the objective. Optimize the lighting conditions, by adjusting exposure times, lamp brightness, aperture and the polarity of the light.
Then, drive the stage to the desired area of the root and focus on the root hairs of interest. To quantify root hair growth over time, set up the software to acquire one image per minute, at the location of interest. In order to visualize plant auto fluorescence or fluorescence markers, minimize the fluorescence exposure time, while still retaining an identifiable fluorescent signal.
Acquire images of the organelles as quickly as the exposure time will allow. Once the seedling has grown for the desired amount of time, remove the device and cover slip from the petri dish. Turn the device upside down and gently peel away the cover slip so that the root stays within the PDMS channel.
Place the PDMS device on a flat reflective substrate, such as, microcoated in gold, and then, mount the device on the AFM specimen holder, root side up. After securing a liquid well attachment on top of the PDMS device, fill it with water to keep the roots hydrated during imaging. Once the specimen has been loaded into the AFM, adjust the z control for the thickness of the PDMS device and drive the cantilever to the region of interest on the root.
Using a camera for guidance, align the laser with the tip of the cantilever and use contact mode imaging to exert minimal force on the root during scanning. Slowly lower the scanning mechanism until the cantilever just makes contact with the sample. Then, adjust the scan size for the desired region and choose a scan speed of one line per second with 256 voltage points per line.
Finally, acquire the scan. The two layer PDMS microfluidic devices described here, have a 200 micrometer high center channel for the main Arabidopsis root and two 20 micrometer high side chambers, to confine the laterally growing root hairs. This ensures that the root hairs are on the same imaging plane, as the main root.
Within the microfluidic platform, root hairs can be quantified on a cellular level, in terms of their length, density and the angularity of their growth from the root. One of the strongest advantages of using a microfluidic platform, is the ability to uniformly add precise concentrations of chemical treatments to the organisms. Here, a seedling is imaged before and after the addition of fluorescent carboxylated polystyrene beads.
The addition of this abiotic treatment, did not disrupt the orientation of optical focus of the seedlings'root hairs. Using fluorescent markers, cytoplasmic streaming can be seen in a root hair. The movement of these three organelles are captured in this cumulative distribution plot.
In addition to optical imaging, this platform also allows for AFM and SEM imaging, due to its ability to be disassembled, without disturbing the root. Following this procedure, other methods including chemical imaging, can be performed in order to answer additional questions like what do chemical profiles around the root look like.
