|Home | About | Journals | Submit | Contact Us | Français|
The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.
Note: Perform all steps preparatory steps under sterile conditions.
The RootChip controller interface for the LabView software platform can be downloaded from our website http://dpb.carnegiescience.edu/technology/rootchip.
The prime purpose of the RootChip is to combine an imaging platform and a perfusion system in a single device with a high level of integration. To demonstrate the manipulation of the microenvironment of roots we flushed the chambers with dark food coloring (1:4 dilution in hydroponic medium) and measured the exchange of fluid within the chambers. At the recommended pressure of 5 psi we measured a full exchange within 10 seconds at a calculated flow rate of approximately 1.5 μl/min (Figure 3).
We also observed root growth of seedlings, in this case grown in the dark and supplied with 10 mM Glucose as an external energy source (Figure 4). Depending on the growth conditions such as light and composition of the medium, plants can be observed in the RootChip for up to three days.
The RootChip has been used to monitor intracellular glucose and galactose levels in roots expressing genetically encoded nanosensors, based on Förster Resonance Energy Transfer (FRET)5-7. Roots in the chip were perfused with square pulses of glucose or galactose solution (Figure 5). The intracellular levels of sugars were monitored and are shown here expressed as a ratio of the intensity of the acceptor fluorophore Citrine to the intensity of the donor ECFP. The rise in ratio indicates accumulation of sugar.
Figure 1. RootChip principle.
Figure 2. Connecting and mounting the RootChip.
Figure 3. Exchange of solutions in the observation chambers. Visualization of the exchange of fluid in an observation chamber using dye solution. The image is an overlay of bright field and false-colored intensity of the dye signal.
Figure 4. On-chip root growth. Observation of a single growing root expressing a fluorescent FRET nanosensor for glucose/galactose over the course of 20h. Time format: hh:mm; scale bar: 100 μm.
Figure 5. Measuring intracellular sugar levels using FRET nanosensors.
The main advantages of the RootChip 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 healthy tissue over a period of several days. Previously, seedlings were grown vertically on gelled media and transferred to a perfusion system immediately before the experiment, which allowed only measuring single roots at a time8. Microfluidic tools have been used for Arabidopsis, but on a low integration level9 or without perfusion control10. The RootChip combines a high level of integration with the ability to automate experiments through precise flow guidance. Another advantage of this platform, characteristic of all microfluidic devices11, is that only minimal amounts of liquid are required to supply the root with the necessary nutrients, even for experiments spanning several days. The RootChip is currently designed as a single-use device, but since production costs of chips are low, the small amounts of consumed reagents makes the chip still very cost-effective.
There are a few critical steps that must be taken to guarantee the health of the seedlings:
The volume in the plastic cones is only 3-4 μl, which will begin to dry when exposed to air. Hence it is critical that the cones are transferred onto the chip quickly and humidity is kept high until the roots have reached the observation chambers, which will supply them with sufficient water. Steps 4.2 to 4.5 should be performed quickly and without interruption to prevent drying of the seedlings.
Steps 3.5 - 3.8 describe the incubation of the chip in liquid media during which the roots grow into the observation chambers. This step may be skipped by mounting the chip into the carrier immediately and starting constant perfusion with growth medium. However, we recommend soaking in growth medium overnight, as it has some advantages: 1) it creates a humid environment so the seedlings are less likely to become desiccated as they grow into the observation chamber; 2) the chip is soaked in liquid, so degassing (step 6.4) will be faster.
It is important to use media with low solute concentrations. More concentrated solutions may precipitate and clog the channels, especially if the chip is used over several days.
Once the device is connected to the air pressure line, flow of medium is controlled by changing hydraulic pressure in the valves. To guarantee proper closure of the micromechanical valves, it is important to choose a control pressure that is about three times higher than the flow pressure. The flow pressure should not exceed 15 psi as the fluid will be pushed out of the root inlets. Higher pressures may also cause delamination of the chip, which renders the chip unusable.
A limitation of the RootChip is that PDMS is porous and hydrophobic. While the material is practically inert to aqueous solutions, it may absorb organic compounds12. This can interfere with a rapid exchange of solutions as organic compounds may leak from the material even when the supply of this compound has been stopped at the inlet. Due to the porosity, using organic solvents may cause swelling of the PDMS12.
We continue to optimize the RootChip and extend its utility, for example with roots of crop plants. We believe that by improving access to the root for treatments and observation, microfluidic tools like the RootChip will open up new dimensions of root research.
No conflicts of interest declared.
We thank Philipp Denninger for help with video preparation and Bhavna Chaudhuri for providing plant lines expressing FRET sensors. This work was supported by grants from the National Science Foundation (MCB 1021677), the Department of Energy (DE-FG02-04ER15542) to W.B.F, the National Institutes of Health, and the Howard Hughes Medical Institute to S.R.Q. G.G. was supported by an EMBO long-term fellowship. M.M. was supported by the Alexander von Humboldt Foundation.