Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Summary

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Abstract

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Introduction

Fine root features increase water and nutrient acquisition for the plant, exploring new soil spaces and increasing the total root surface area. The turnover of these fine root features plays a major role in stimulating the underground food chain¹ and the number of fine roots in certain plant species is expected to double under elevated atmospheric carbon dioxide². Fine roots are generally defined as those smaller than 2 mm in diameter, although new definitions advocate for characterizing fine roots by their function³. Like many fine roots, root hairs provide the function of uptake and absorption but occupy a much smaller space with diameters on the order of microns. Because of their small size, root hairs are difficult to image in situ and are often overlooked as a part of the overall root architecture in field scale experiments and models.

Ex terra root hair studies, such as from seedlings grown on agar plates, have provided the scientific community with valuable information on cellular growth and transport^4,5. While agar plates allow root systems to be imaged non-destructively and in real time, they do not offer high environmental control for the addition of experimental treatments such as nutrients, plant hormones, or bacteria. An emerging solution to facilitating high resolution imaging while also affording dynamic environmental control has been the advent of microfluidic platforms for plant studies. These platforms have enabled the non-destructive growth and visualization of several plant species for high throughput phenotyping^6,7,8,9, isolated chemical treatments¹⁰, force measurements^11,12, and the addition of microorganisms¹³. Microfluidic platform designs have focused on the use of single open space fluidic layers in which the roots may propagate, permitting the root hairs to drift in and out of optical focus during growth or treatment.

Here we present a procedure for developing a two-layer microfluidic platform using photo and soft-lithography methods that builds upon previous plant-on-a-chip designs by confining the seedling root hairs to the same imaging plane as the main root. This allows us to track root hair development in real time, at high resolution, and throughout the experimental treatment process. Our culturing methods allow Arabidopsis thaliana seedlings to be germinated from seed within the platform and cultured for up to a week in a hydrated and sterile environment that does not require the use of syringe pump equipment. Once the time-lapse imaging experiment has concluded, the platform presented here can be opened without disturbing the position of the finer root features. This allows the use of other high resolution imaging methods. Here we provide representative results for the quantification and visualization of root hair morphology in this platform by optical, scanning electron microscopy (SEM), and atomic force microscopy techniques (AFM).

Protocol

1. Two-layer Platform Fabrication

1. Fabrication of multilayer masters
   1. Spin coat epoxy-based negative photoresist (~63.45% solids, 1,250 cSt) according to the manufacturer's specifications (2,000 rpm for 45 s) onto a 4 inch diameter silicon wafer to obtain the desired height of 20 µm for the first design layer.
   2. Soft-bake the resist coated wafers for 4 min at 95 °C. Allow wafer to cool for 5 min. Expose the wafer to UV light for 15 s (~150 mJ/cm² at 365 nm) through a photomask in a UV contact aligner to define the geometry of the bottom layer.
   3. Post-exposure bake the wafer for 5 min at 95 °C. Without developing, return the wafer to the spin coater and spin on a second layer of epoxy-based negative photoresist (~76.75% solids, 80,000 cSt) at 3,000 rpm to achieve a second layer height of 150 - 200 µm.
   4. Allow the wafer to rest for 5 min before transferring to a 95 °C hotplate for 45 min. After baking on the hotplate, remove the wafer and allow the resist to cool and harden at room temperature for another 5 min on a flat level surface. Align and expose the wafer to the second layer photomask for 30 s in a UV contact aligner (dose of approximately 300 mJ/cm²).
   5. Perform a post-exposure bake by placing the wafer on a hotplate for 15 min at 95 °C, and then allow the wafer to cool on a flat level surface for 5 min before developing.
   6. Develop both resist layers simultaneously by placing the wafer into a plastic dish and submerging the wafer in the appropriate developer (see Materials Table). Gently rock the dish occasionally to wash fresh developer over the wafer.
   7. After 17 min, rinse the wafer with isopropanol (IPA). If a white film appears, continue to iterate between rinsing the wafer with developer and IPA until the film disappears. Dry the patterned silicon wafer with nitrogen.
2. Polydimethylsiloxane Soft-lithography
   1. Expose the silicon wafer to an air plasma for 30 s in a plasma cleaner set on high (see Materials Table) to clean. Silanize the wafer with trichloro (1H,1H,2H,2H-perfluoro-n-octyl) silane in a chemical hood on a hotplate below the flashpoint of the silane (85 °C) for 2 h.
   2. Pour a 10:1 ratio of polydimethylsiloxane (PDMS) to curing agent onto the silicon master wafer. Degas the mixed PDMS in a vacuum chamber and then cure the polymer in a 70 °C oven for 1 h or until the PDMS is fully cured.
   3. Using a scalpel, cut the PDMS devices and peel them from the silicon master wafer. Use a 1.5 mm biopsy punch to create seed and treatment inlets, punching the seed inlet at a 45° angle to encourage root growth into the main channel.
   4. Use clear adhesive tape to remove debris from the PDMS device and place the device design side down on a glass coverslip. Autoclave the assembled devices.

2. Planting Devices

1. Arabidopsis thaliana seed preparation
   1. Surface sterilize A. thaliana seeds in a microfuge tube with a solution of 30% commercially available bleach diluted in de-ionized water and 0.1% Triton X detergent for 7 min. Wash seeds 4 times with sterile water.
   2. Stratify seeds by refrigerating microfuge tube overnight or up to a week at 4 °C.
2. Device Preparation
   1. Place sterilized devices in a vacuum degassing chamber to remove air from the gas permeable PDMS and fluidic channels to improve ease of filling.
   2. Remove the devices from the vacuum chamber and immediately submerge the devices in a Petri dish filled with liquid ¼ strength plant based media at pH 5.7.
   3. Using a pipette, pull liquid through the inlets to fill the device. Make sure no air bubbles remain within channels by visual inspection.
   4. Transfer individual devices to new dry Petri dishes. Pour or pipette hot agar around the device until the agar level is almost flush with the top of the PDMS device. Allow agar to solidify.
      NOTE: Devices are now ready for planting seeds or can be stored at 4 °C until needed.
3. Planting seeds within devices
   1. In a sterile environment, transfer one sterilized seed to the inlet of each device using a small pipette.
   2. Cover the Petri dish with wax film (see Materials Table) and place in a light/dark cycling growth chamber or windowsill at room temperature. Orient the Petri dish so that the devices are vertical and gravity will encourage the roots to grow through the channel.

3. Treatment

1. Experimental Treatments
   1. At the desired time in the seedling's development, add the experimental treatment to the seedling by adding a prescribed amount of the treatment to each of the 8 side ports via pipette.
   2. Reseal the Petri dish and return to growth chamber or windowsill.
   3. Proceed with imaging samples at the desired time to capture individual time points or begin time lapse imaging.

4. Optical Imaging

1. Lower Resolution (4 - 20X) Imaging
   1. Place the entire Petri dish containing the device and seedling under an inverted bright field microscope for lower resolution (4 - 20X) bright field or differential interference contrast (DIC) imaging. Optimize lighting conditions to elucidate morphological features of interest by adjusting exposure times, lamp brightness, aperture, and the polarity of light. Drive the stage to the desired area of the root and focus on the root or root hair(s) of interest.
   2. Acquire a single time point or a time series of images. To visualize the growth of root hairs, image growing root hairs once per min. To visualize the growth of the main root, acquire one image every 30 min. Return the Petri dish and the seedlings to growth chamber or windowsill in a vertical position after imaging is complete.
2. Higher Resolution (20 - 63X) Imaging
   1. Once the seedling has grown for a desired time, use forceps to remove coverslip and device from the agar. Clean off the bottom of the coverslip using an ethanol wetted laboratory tissue.
   2. Apply the recommended immersion media to the objective as suggested by the microscope lens manufacturer. Place the coverslip down on the microscope stage and 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 light. Drive the stage to the desired area and focus on root hair(s) of interest.
      NOTE: DIC is needed to visualize cytoplasmic streaming, and fluorescence markers are needed to visualize organelle movement.
   3. To quantify root hair growth over time, acquire one image per min. To visualize organelle movement, minimize the fluorescence exposure time while still retaining an identifiable fluorescence signal from the organelles. Acquire images of the organelles as quickly as the exposure time allows. For longer time-lapse imaging, use a live-cell imaging stage incubator to maintain ambient temperature and moisture.
      NOTE: A 63X oil objective is recommended for imaging root hair organelles.

5. Non-Optical Imaging

1. Device preparation
   1. Once seedling has grown for desired time, remove the device and coverslip from the Petri dish.
   2. Turn device upside down and gently peel away the coverslip so that the root stays within the PDMS channel.
   3. Proceed to use the PDMS device as a substrate for the root in higher resolution atomic force or scanning electron microscopy.
2. Scanning Electron Microscopy
   1. Deposit a thin (~20 nm) layer of chromium onto the root and surrounding PDMS using a Dual Gun Electron Beam Evaporation Chamber.
   2. Transfer the root and PDMS device to a scanning electron microscope chamber.
      NOTE: Imaging conditions, i.e. voltage, current and working distance will need to be optimized to achieve the desired resolution for a given application.
3. Atomic Force Microscopy (AFM)
   1. Mount the PDMS device root side up on an AFM specimen holder. To increase contrast in the camera and more easily distinguish the root from the PDMS, place the PDMS device on a flat reflective substrate (such as mica coated in gold) before mounting the device on the AFM specimen holder.
   2. Secure a liquid well attachment on top of the PDMS device and fill it with water to keep the roots hydrated during imaging.
   3. Load the specimen holder 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.
   4. Align the laser with the tip of the cantilever. For best results with contact mode imaging, use cantilevers with spring constants of 0.01 or 0.03 N/m to exert minimal force on the root during scanning.
   5. Slowly lower the scanning mechanism until the cantilever just makes contact with the sample. Adjust the scan size for the desired region and choose a scan speed of 1 line/s with 256 voltage points per line. Acquire the scan.

Results

The two-layer PDMS microfluidic devices described here have a 200 µm high channel for the main Arabidopsis root and a 20 µm high chamber to confine laterally growing root hairs (Figure 1A). This design may be used for plant species with similar root diameters as Arabidopsis thaliana and can be readily modified to accommodate species of different sizes. The design incorporates an inlet for the plant as well as 8 side inlets for any d...

Discussion

The method described in this article for creating a plant-on-a-chip platform is unique in that the two-layer design confines the root hairs to a single imaging plane and the platform may be deconstructed and used as a substrate for high resolution non-optical imaging. Using high-resolution non-optical imaging can provide valuable information about the plant tissue that could not be obtained from optical imaging alone. For example, AFM imaging can provide force measurements to calculate the elasticity of root tissues duri...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon Wafer	WRS Materials	100mm diameter, 500-550um thickness, Prime, 10-20 resistivity, N/Phos<100>
Quintel Contact Aligner	Neutronix Quintel Corp	NXQ 7500 Mask Aligner
Fluorescent Microscope	Nikon	Eclipse Ti-U
laboratory tissue	Kimberly Clark	Kimwipe KIMTECH SCIENCE Brand, 34155
Negative Photoresist Epoxy	Microchem	SU-8 2000s series
Photoresist developer	Microchem	Su-8 developer
trichloro(1H,1H,2H,2H-perfluoro-octyl)silane	Sigma Aldrich		use in chemical hood
Air Plasma Cleaner	Harrick Plasma
PDMS	Dow Corning	Sylgard 184 Silicone elastomer base
PDMS curing agent	Dow Corning	Sylgard 184 Silicone elastomer curing agent
Dessicator	Bel-Art	F42010-000
Scalpel	X-acto knife
Biopsy Punch	Ted Pella	15110-15
Adhesive tape	Staples	Invisible Tape
Microfuge tube	Eppendorf
Triton X	J.T.Baker XI98-07
Bleach	Chlorox	concentrated
Plant-Based Media	Phyto Technology Laboratories	M524
Agar	Teknova	A7777
Wax film	Parafilm
microscope	Olympus	IX51
Atomic Force Microscope	Keysight Technologies	5500 PicoPlus AFM
Petri dish	VWR
Scanning Electron Microscope	JEOL	7400
Dual Gun Electron Beam Evaporator	Thermionics	Custom Dual Electron Gun Evaporation System