This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).
This work was supported in part by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov). The fabrication of the microfluidic platforms was carried out in the Nanofabrication Research Laboratory at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility. JAA is supported by an NSF graduate research fellowship DGE -1452154

1. Two-layer Platform Fabrication
<ol>
	<li>Fabrication of multilayer masters
	<ol>
		<li>Spin coat epoxy-based negative photoresist (~63.45% solids, 1,250 cSt) according to the manufacturer&#39;s specifications (2,000 rpm for 45 s) onto a 4 inch diameter silicon wafer to obtain the desired height of 20 &#181;m for the first design layer.</li>
		<li>Soft-bake the resist coated wafers for 4 min at 95 &#176;C. Allow wafer to cool for 5 min. Expose the wafer to UV light for 15 s (~150 mJ/cm2 at...

<ol>
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Vis. Exp. (65), (2012).</li><li>Jiang, H., Xu, Z., Aluru, M. R., Dong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chip+for+high-throughput+phenotyping+of+Arabidopsis.">Plant chip for high-throughput phenotyping of Arabidopsis.</a> Lab Chip. 14 (7), 1281 (2014).</li><li>Busch, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+device+and+computational+platform+for+high-throughput+live+imaging+of+gene+expression.">A microfluidic device and computational platform for high-throughput live imaging of gene expression.</a> Nat. Methods. 9 (11), (2012).</li><li>Meier, M., Lucchettta, E., Ismagilov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Stimulation+of+the+Arabidopsis+thaliana+Root+using+Multi-Laminar+Flow+on+a+Microfluidic+Chip.">Chemical Stimulation of the Arabidopsis thaliana Root using Multi-Laminar Flow on a Microfluidic Chip.</a> Lab Chip. 10 (16), 2147-2153 (2010).</li><li>Ozoe, K., Hida, H., Kanno, I., Higashiyama, T., Notaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+characterization+method+of+plant+root+adaptability+to+soil+environments.">Early characterization method of plant root adaptability to soil environments.</a> Proc. of 28th IEEE Interntl. Conf. Micro. Electro Mech. Syst. , (2015).</li><li>Sanati Nezhad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+platforms+for+plant+cells+studies.">Microfluidic platforms for plant cells studies.</a> Lab on a chip. , 3262-3274 (2014).</li><li>Parashar, A., Pandey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-in-chip:+Microfluidic+system+for+studying+root+growth+and+pathogenic+interactions+in+Arabidopsis.">Plant-in-chip: Microfluidic system for studying root growth and pathogenic interactions in Arabidopsis.</a> App. Phys. 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Exp. Bot. 62 (1), 59-68 (2011).</li><li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies.</a> Electrophor. 24 (21), 3563-3576 (2003).</li><li>Millet, L. J., Stewart, M. E., Sweedler, J. V., Nuzzo, R. G., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+culturing+primary+mammalian+neurons+at+low+densities.">Microfluidic devices for culturing primary mammalian neurons at low densities.</a> Lab chip. 7 (8), 987-994 (2007).</li><li>Nelson, B. K., Cai, X., Nebenf&#252;hr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolored+set+of+in+vivo+organelle+markers+for+co-localization+studies+in+Arabidopsis+and+other+plants.">A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.</a> Plant J. 51 (6), 1126-1136 (2007).</li><li>Talbot, M. J., White, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+and+cell+outline+imaging+in+plant+tissues+using+the+backscattered+electron+detector+in+a+variable+pressure+scanning+electron+microscope.">Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope.</a> Plant Methods. 9 (1), 40 (2013).</li></ol>

The authors have nothing to disclose.

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...

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 transport4,5. 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 phenotyping6,7,8,9, isolated chemical treatments10, force measurements11,12, 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).

<table width="810">
	<tbody>
		<tr height="72">
			<td height="72" style="width:209px;height:72px;">Silicon Wafer</td>
			<td style="width:191px;">WRS Materials</td>
			<td style="width:283px;">100mm diameter, 500-550um thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="width:209px;height:32px;">Quintel Contact Aligner</td>
			<td style="width:191px;">Neutronix Quintel Corp</td>
			<td style="width:283px;">NXQ 7500 Mask Aligner</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="width:209px;height:32px;">Fluorescent Microscope</td>
			<td style="width:191px;">Nikon</td>
			<td style="width:283px;">Eclipse Ti-U</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="width:209px;height:30px;">laboratory tissue</td>
			<td style="width:191px;">Kimberly Clark</td>
			<td>Kimwipe KIMTECH SCIENCE Brand, 34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Negative Photoresist Epoxy</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:283px;">SU-8 2000s series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Photoresist developer</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:283px;">Su-8 developer</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">trichloro(1H,1H,2H,2H-perfluoro-octyl)silane</td>
			<td style="width:191px;">Sigma Aldrich</td>
			<td></td>
			<td>use in chemical hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Air Plasma Cleaner</td>
			<td style="width:191px;">Harrick Plasma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">PDMS</td>
			<td style="width:191px;">Dow Corning</td>
			<td>Sylgard 184 Silicone elastomer base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">PDMS curing agent</td>
			<td style="width:191px;">Dow Corning</td>
			<td>Sylgard 184 Silicone elastomer curing agent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Dessicator</td>
			<td style="width:191px;">Bel-Art</td>
			<td>F42010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Scalpel</td>
			<td style="width:191px;">X-acto knife</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Biopsy Punch</td>
			<td style="width:191px;">Ted Pella</td>
			<td>15110-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Adhesive tape</td>
			<td style="width:191px;">Staples</td>
			<td>Invisible Tape</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Microfuge tube</td>
			<td style="width:191px;">Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Triton X</td>
			<td style="width:191px;">J.T.Baker XI98-07</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Bleach</td>
			<td style="width:191px;">Chlorox</td>
			<td>concentrated</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">Plant-Based Media</td>
			<td style="width:191px;">Phyto Technology Laboratories</td>
			<td>M524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Agar</td>
			<td style="width:191px;">Teknova</td>
			<td>A7777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Wax film</td>
			<td style="width:191px;">Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">microscope</td>
			<td style="width:191px;">Olympus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Atomic Force Microscope</td>
			<td style="width:191px;">Keysight Technologies</td>
			<td>5500 PicoPlus AFM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Petri dish</td>
			<td style="width:191px;">VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>7400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">Dual Gun Electron Beam Evaporator</td>
			<td>Thermionics</td>
			<td>Custom Dual Electron Gun Evaporation System</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging the root hair morphology of arabidopsis seedlings in a two-layer microfluidic platform

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.

The two-layer PDMS microfluidic devices described here have a 200 &#181;m high channel for the main Arabidopsis root and a 20 &#181;m 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...

Watch this Scientific Journal Video about Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform at JoVE.com

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

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.

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

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 ...

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.

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JoVE Journal

Bioengineering

8.4K Görüntüleme.  University of Tennessee. 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 imposs...