Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

In order to provide maximal Yields, plants need to be provided with a suite of nutrients, nutrient deficiencies, stresses such as cold or extreme heat, drought, or pathogens cause massive losses in crop yield every year. The origin of many of these problems often lies underground. The root is the physical anchor of the plant, but it is also the organ that is responsible for water uptake and for the uptake of mineral nutrients such as nitrogen, phosphorous sulfate, and many trace elements.

If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the roots develops, how it takes up this wide spectrum of nutrients and how it interacts with symbiotic and pathogenic organisms. To do this, we need to be able to explore roots at the microscopic level For obvious reasons. Studying the biology of roots has always been more challenging than the study of aerial parts of the plant.

Since roots are usually hidden underground, they're not easily accessible for microscopic studies. Removal from soil causes severe damage to the root system and is thus not a good way to study their behavior. One solution to make roots more accessible is to grow them on jeed or is shown in this example in hydroponic media.

Growing plans in jelled media or in hydroponic media has been very successfully used in many root studies, but with these methods, it's still very difficult to study roots in microscopic detail and over long periods of time to get as close as possible to the growing root and avoid any physical stress from preparation for imaging, we build a platform that allows us to grow and image roots, and at the same time allows us to control and modify the microenvironment of the roots with very high precision and speed. This platform we called the root chip. The root chip is a microfluidic device fabricated from PDMS, a silicone-based organic polymer.

The chip features observation chambers for the growth and imaging of roots from a rabbit opsis seedlings. The seeds first germinate in plastic cones fabricated from plastic pipette tips, which we fill with solid media. The root tip then grows through the medium and reaches the chamber where it experiences a continuous flow of liquid medium, keeping the conditions in the chamber.

Constant micro mechanical valves developed by Steve Quake's lab at Stanford University here is shown in red control. The flow since the chip is mounted on a transparent cover glass as typically used for microscopy, all processes in the observation chamber can be monitored on an inverted microscope. The bifurcated channel structure that guides the fluid flow can be seen under the microscope.

The root chip consists of two layers, a control layer containing the valves in a flow layer containing the channels through which growth media test solutions flow to the observation chambers. The volume of these chambers is approximately 400 nanoliters. This means that only very small amounts of solution are needed and conditions can be changed very rapidly.

This protocol describes how live roots of eight or Arabidopsis seedlings are prepared for parallel microscopic imaging on the root chip and observed for up to three days begin by filling a 10 millimeter Petri dish with growth medium containing 1%agar while the medium is still liquid. Fill 10 microliter pipette tips with five microliters of medium from the Petri dish using a multi-channel pipette. The filled tips are stored in the pipette tip box until the medium is solid.

Then cut to four millimeter long plastic cones and placed upright into a Petri Dish containing solid growth medium. After surface sterilization, single seeds are placed on top of the media filled cones. The dish is then sealed with micropore tape and the plate is stored at four degrees to Synchronize germination.

After three days, the plates are transferred to a growth cabinet to start germination. Our growth conditions in this experiment are 23 degrees. At a 16 hour highlight, eight hour darkness cycle between five and seven days after germination, the seedling should be ready for transfer to the root chip seedling health root length, and if applicable, Expression of a fluorescent marker should be checked under a dissecting microscope.

As soon as the root tips of the seedlings Reach the bottom outlet of the plastic cone individual seedlings are marked for transfer onto the chip. 10 or so Plant seedlings should be selected in case one gets damaged during transfer to sterilize the root chip. For long-term experiments, the device is wrapped in tissue, placed in a glass Petri dish and autoclave.

This chip has just been autoclave before. The experiment after Cooling down the chip is overlaid with liquid growth media. The chip should be completely covered, but the fluid level should be no higher than three millimeters over the chip surface.

A 20 microliter pipette is used to pull media through the Root inlet and chamber outlet. This fills the observation chamber. With media selected plastic cones are now plugged into the root chip.

The cone Should fit snugly in the inlets. Since the root chip is mounted on a thin layer of optical glass, one must be careful not to apply Too much pressure to the chip. During this step, the chip is now prepared for an overnight incubation within the Liquid media to prevent the chip from floating.

Two glass slides, one cut in half are placed onto the chip. A magnetic stir bar is added and the dish is closed. The assembly is now transferred to a magnetic stir, which will gently agitate the medium to facilitate root growth towards the outlets.

The root chip's. Inlets are punched at an angle to further support growth in the desired direction. Slightly tilt the assembly by lifting the side of the chip that is opposite of the outlets.

The chip is illuminated with ring lamp Connected to a timer switch to maintain the light dark rhythm. After the overnight incubation, liquid growth media Is prepared in a sealable pressurable vial. The following steps should be performed quickly and without interruption to prevent drying of the seedlings.

The chip is now removed from the liquid medium and placed upside down into the bottom aperture of The chip carrier, which is also inverted. The chip must be oriented so that the side Containing the control layer inlets is facing the side of the pressure line. Two big connectors in the carrier sidewall.

The cover glass on the bottom of the chip is dried by Gently blotting with tissue paper and affixed to the carrier. With tape, the whole assembly is then inverted. The tubing connectors are filled with water using a syringe, and each tubing connector is plugged into the corresponding inlet.

On the chip. Tubing is plugged into the media and solution. Vials and air is then removed from the lines by applying pressure to the solution bile with syringe of air, the carrier is covered with transparent plastic.

To maintain high humidity in the assembly, the carrier is placed onto the microscope stage. It should fit exactly into the Notches of the stage. The chip valves, as well as the flow of medium through the chip are controlled by air pressure.

Two lines with regulators are branched off of a main pressure line. One is used to control the fluid flow and the other is connected to solenoid air valves. These valves are operated from the computer via the USB valve controller and are responsible for actuating the valves on the chip.

Both pressure regulators should be closed before the chip is connected. Tubing, connectors and solution vials are now attached to the corresponding pressure lines. A few milliliters of water are added to the reservoirs of the carrier.

This step may need to be repeated over the course of longer experiments. To keep the humidity high, keep the volume load to minimize the potential amount of liquid that could be spilled onto the microscope. The ring light is moved into position over the chip to Maintain the light dark cycle until the experiment begins.

Valves on the chip are closed by applying pressure in This case by opening the solenoid air valves. The lab view interface allows control of the valves by clicking the button below the valve number. Bright green indicates application of pressure and closing of a chip valve.

This scheme illustrates the organization of the valving system while valves four through eight act as single valves valve zero through three act. In groups with this system, an individual chamber can be addressed by activating a combination of valves, for example, to guide the fluid flow exclusively to the third chamber from the top valves zero, three and four must be closed. In addition, the valve six, seven, and eight control which solution is used for flushing the inlets A, B, and C now activate all three solution inlet valves to close them.

The controller interface features a feedback loop, which allows monitoring of the system's status. This feature may be activated by clicking on the read back button. The pressure regulator for the control layer is open first and set to 15 PSI.

Then the regulator for the flow layer is open and set to five PSI. The inlet valve for the growth media of choice is opened and the chambers are flushed with media flow paths should be checked under the microscope. In most cases, air is trapped in the channels and must be removed.

Additionally, the channels of the control air still contain air that must be forced out and replaced by the water from the tubing connectors. This process is called dead end filling. Both tasks are achieved by flushing each of the eight chambers several times until all air is forced from the channels into the PDMS.

The system can be programmed to automate experiments. Such routines may also be used to degas the chip. The prime purpose of the root chip is to combine an imaging platform And a profusion system in a single device.

To demonstrate the manipulation of the microenvironment of roots, we flushed the chambers with dye and measured the exchange of fluid within the chambers. At the recommended pressure of five PSI, we measured a full exchange within 10 seconds at a calculated flow rate of approximately 1.5 microliters per minute. We also observed root growth of seedlings in this case grown in the dark and supplied with 10 millimolar glucose as an external energy source.

Depending on the growth conditions such as light and composition of the medium, the plants can be observed in the root chip for up to three days. This system has been used to monitor intracellular glucose and galactose levels in roots expressing genetically encoded nanosensors. These sensors based on first or resonance energy transfer or fret were developed in the Fromer lab.

For this experiment, roots in the chip were perfused with square pulses of glucose or galactose solution. The intracellular levels of sugars were monitored and are shown here, expressed as a ratio of the intensity of the acceptor fluoro four citrine to the intensity of the donor ECFP to the left. We observe the amount of sensors citrine in the middle or ratio metric image of the root tip, and to the right, we trace the amount of sugar as a response to three repeated square pulses of glucose in green and galactose shown in red.

The rise in ratio indicates an accumulation Of sugar. The main advantages of the root chip over conventional growth methods are the minimally invasive preparation for microscopy, the ability to reversibly and repeatedly alter the root environment and the capacity for continuous observation of developmentally competent and physiologically health tissue over a period of days. Another really important advantage is that only minimal amounts of liquid are needed to supply the roots with all the necessary nutrients over time.

This makes the root chipp very cost effective, especially when expensive reagents are applied. We continue to optimize the root chipp and extend its usability because we believe that by making this important organ better accessible for treatments and observation, microfluidic tools like the root chip will potentially open up new dimensions of plant research. More information on how to build a root chip system can be found on our website.

Chips can be ordered from the Stanford Foundry.