We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).

1. Plant Growth
<ol>
	<li>Surface sterilize seeds.
	<ol>
		<li>Sterilize seeds (up to 30 mg) by wet or dry method. As an example, for dry sterilization method, add 1 ml glacial HCl 37% in 50 ml bleach, in a glass beaker in a closed desiccator for at least 4 hr and up to overnight. Perform this step in a chemical hood.</li>
	</ol>
	</li>
	<li>Spread the sterile seeds on plates with 0.8% plant agar in 0.5x Murashige &#38; Skoog (MS) medium. Wrap plates with Parafilm or porous tape and cover with aluminum foil.</li>
	<li>Store the plates at 4 &#176;C for 1 - 4 days to promote uniform germination.</li>
	<li>Transfer the plates to a growth chamber suitable for growing Arabidopsis seedlings. Place the plates vertically to maintain root growth on top of the agar medium and to prevent penetration.</li>
	<li>Transfer seedlings to fixative 7 days after germination (DAG).</li>
</ol>
2. Fixation
<ol>
	<li>Prepare 50 ml Richardson Solution by combining equal volumes of 1% Borax (buffer agent), 1% Methylene blue and 1% Azure (histological dyes) in double distilled water. Filter through a Syringe Driven 0.22 &#956;m PVDF Filter Unit before use.</li>
	<li>Prepare 50 ml of fixative: 1.25% Glutaraldehyde in 0.05 M Sodium Cacodylate (buffering agent). 
	Note: These materials are toxic and must be handled in a chemical hood.</li>
	<li>Add 100 &#181;l of Richardson solution (see 2.1) to the fixation solution (see 2.2) to obtain the final fixation solution.</li>
	<li>Mark each well of a 6-well plate with the corresponding sample name.</li>
	<li>Place 2 - 3 ml final fixation solution (see 2.3) into the wells, so that the seedlings, once transferred, will be fully covered in liquid.</li>
	<li>Carefully transfer, using forceps, up to 10 seedlings to each well. Seal the plates using Parafilm. Store at 4 &#176;C, in darkness, for at least one night, and up to one week.</li>
</ol>
3. Dehydration
<ol>
	<li>Dehydrate seedlings. Transfer them between wells of new 6-well plates, containing increasing concentrations of ethanol, as listed here: 10% ethanol for 15 min; 30% ethanol for 15 min; 50% ethanol for 15 min; 70% ethanol for 15 min; 85% ethanol for 15 min; 95% ethanol for 15 min; 95% ethanol at 4 &#176;C, overnight, at minimum. Handle seedlings by carefully grasping them by their cotyledons, preferable with plastic forceps.</li>
</ol>
4. Infiltration&#160;
<ol>
	<li>Prepare infiltration medium. Mix the contents of 1 bag of the historesin kit activator with 50 ml Basic resin liquid kit in a small glass bottle. Stir with magnet for 20 min. Infiltration medium may be kept at 4 &#176;C for a few weeks.</li>
	<li>Mark each well of a new 6-well plate with the corresponding sample name and fill with 2 - 3 ml of infiltration medium.</li>
	<li>Transfer the seedlings from the 95% ethanol solution to the infiltration medium using forceps. Make sure the seedlings are fully covered by the medium.</li>
	<li>Store at 4 &#176;C for at least 4 days, to ensure maximum penetration into the plant tissues.</li>
</ol>
5. Block Preparation
<ol>
	<li>Place small labels, pencil-marked with sample name, inside each well of embedding molds.</li>
	<li>Prepare embedding medium by adding 1 ml hardener to 15 ml infiltration medium (see 4.1). Do not attempt to prepare a volume smaller than 15 ml, to avoid polymerization issues.</li>
	<li>Fill half of each well in the mold with 200 &#181;l embedding medium and cover with an overhead film piece cut to the mold shape, but to a size 1 mm larger on each side. Incubate at room temperature for at least 2 hr, to allow polymerization. Do not exceed 5 hr.</li>
	<li>Prepare an additional batch of embedding medium (see 5.2) and keep on ice, to avoid polymerization while root tips are being arranged in the mold.</li>
	<li>Cut each root tip under a dissecting scope using scalpel and very carefully position it: vertically for transverse sections or horizontally for longitudinal section, at about 2 - 3 mm from the mold periphery. Fill the mold completely with blocking solution and cover with an overhead film. Note that the blocking solution will shrink slightly once it is polymerized.</li>
	<li>Keep at room temperature for 2 hr, at minimum.</li>
	<li>To dry the blocks, place in a box with dry silica gel and leave at room temperature overnight, at minimum.</li>
</ol>
6. Sectioning
<ol>
	<li>Prepare glass knives from glass rods with a knife maker. Use the glass knives for tissue sectioning. Cut the glass rod into a square and then cut the square diagonally to produce two knives, using a diamond scoring tool.</li>
	<li>Section blocks into 2 - 3 &#181;m width slices, using a sectioning machine. Place slices on a glass slide, on top of distilled water droplets and place the slide on a heat block set to low heat (50 - 60 &#176;C), until water evaporates.</li>
</ol>
7. Slide Preparation for Polarized Microscope
<ol>
	<li>Dilute Richardson solution (see 2.1) to 0.2% of the original solution and use 1 ml of it to cover the slices. Place on the heat block for 5 sec and then wash with distilled water.</li>
	<li>Dry the slide on a hot plate. Observe under a dissecting scope and mark the back of the slide with a marker, to indicate the position of the slices of interest, to facilitate their later identification under the microscope.</li>
	<li>Add 100 &#181;l of mounting medium and cover with a cover slip.</li>
</ol>
8. Image Acquisition
<ol>
	<li>Place the covered slide containing the root sections under a light microscope equipped with Polscope system suitable to obtain retardance information and a polarizer/interference optic filter (Figure 1 A-D).</li>
	<li>Select magnification that allows clear visualization of the entire sample (e.g., 40X in this study) and focus the microscope on an empty field that contains block section but no root section. Try to find a clean region with no blemishes. Use this region to set the background.</li>
	<li>Set the imaging system and software parameters: Set range to 17 nm. Apply &#34;Auto Exposure&#34; and obtain background image from the empty field (see 8.2). One background image is used per experiment.</li>
</ol>
9. Polscope Analysis of Cellulose Microfibril Orientation
<ol>
	<li>Analysis of longitudinal sections.
	<ol>
		<li>Place a slide containing longitudinal sections under the microscope and obtain a retardance image of it. Focus the microscope on a root section of interest. Provide a new file name.</li>
		<li>Capture an abrio image (image with retardation information) through the &#34;Live Abrio&#34; window. Be sure to capture a section that includes the cell wall, which can be identified by the stained cellulose running along the periphery of the cell, as shown in Figure 2C.</li>
	</ol>
	</li>
	<li>Image analysis.
	<ol>
		<li>Double-click on the relevant image in the image stack. Select &#34;Orientation Pseudo color&#34; in the &#34;Display Settings&#34; tab.</li>
		<li>To estimate the cellulose microfibril angle, match the corresponding shade of the cell wall to that in the color wheel. 
		NOTE: The color wheel in Figure 2C reflects the slow axis of light lying along the microfibril which indicates the direction along the long axis of the microfibrils. The angle of cellulose microfibrils is the angle indicated by the color and the long axis of the cell. For example, the elongating cell in the inset shows cyan color, tilted by about 90&#176; to the long axis of the cells.</li>
		<li>To quantify the average microfibril angle in the cell wall, use &#34;Retardance and Azimuth Stamp&#34; tool from the toolbar of the software. While selecting the tool, point and click on the region of interest in the obtained image. 
		NOTE: This will result in the display of the angles (azimuth) in degrees (Figure 2C).</li>
		<li>In an alternative open access polarized light image analysis software, lunch &#34;Pol-Analyzer&#34; plugin from the plugin menu. In the new opened window, select the &#34;Birefringence&#34; tab and open and then save the background file and the image file.</li>
		<li>In the image window, select &#34;Specs.&#34; and adjust the &#39;ROI pixel size&#39; (use 5 pixels for this study). In the &#34;Specs.&#34; window, select &#34;show values in Result table&#34;.</li>
		<li>Use the Polygon tool to mark the region of interest in the image window. Select &#34;Orientation Lines&#34; to obtain the orientation of the cellulose microfibril. 
		NOTE: This will result in the display of the angles (azimuth) in degrees. 
		NOTE: The average angle calculated by one of the two methods should be then normalized to the positioning of the root section along the slide. To correct for deviation from the horizontal positioning of the whole root section along the slide, quantify the average microfibril angle of the cell wall flanking the cell along its long axis (that is, perpendicular to the section and along the root growth axis, Figure 2C). This angle is 180&#176; when the root section is accurately positioned along the slide. For calculating the microfibril angle, subtract the average measured microfibril angle of the flanking cell wall from 180&#176;. Add the resulted difference to the average measured microfibril angle of the cell wall that lies flat on the slide.</li>
	</ol>
	</li>
</ol>
10. Polscope Analysis of Crystalline Cellulose Accumulation
<ol>
	<li>Use the transverse (cross) sections. Note that crystalline cellulose accumulation can only be compared between cells when their cellulose microfibrils are aligned at a similar angle (as determined in 9).</li>
	<li>Double-click on the relevant image in the image stack. Select &#34;Pseudo color&#34; in the &#34;Display Settings&#34; tab.</li>
	<li>To measure retardance. Zoom-in on the image. Select &#34;Region&#34; and mark all regions of interest. Move to the &#34;measurements&#34; tab and &#34;Copy to Clipboard&#34; all values.
	<ol>
		<li>Transfer the data to Excel and extract required information (mean region of retardance). Normalize the data. 
		NOTE: To account for differences in thickness between slices, express the values relative to a specific cell wall edge in the slide. To normalize, divide the average retardance corresponding to the cell wall of interest by the average retardance of a different cell wall in the same image. For example, the outer epidermal cell wall was normalized to the inner cortical cell wall in our study.</li>
	</ol>
	</li>
	<li>Repeat measurements for at least 3 sections per root, with a minimum of 3 roots per sample.</li>
</ol>

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+plant+cell+wall.">Growth of the plant cell wall.</a> Nat Rev Mol Cell Biol. 6, 850-861 (2005).</li><li>Wolf, S., Hematy, K., Hofte, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control+and+cell+wall+signaling+in+plants.">Growth control and cell wall signaling in plants.</a> Annu Rev Plant Biol. 63, 381-407 (2012).</li><li>Mazeau, K., Heux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Dynamics+Simulations+of+Bulk+Native+Crystalline+and+Amorphous+Structures+of+Cellulose.">Molecular Dynamics Simulations of Bulk Native Crystalline and Amorphous Structures of Cellulose.</a> J. Phys. Chem. B. 107, 2394-2403 (2003).</li><li>Zhao, H., 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=Studying+cellulose+fiber+structure+by+SEM,+XRD,+NMR+and+acid+hydrolysis.">Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis.</a> Carbohydr Polym. 68, 235-241 (2007).</li><li>Fujita, M., 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=Cortical+microtubules+optimize+cell-wall+crystallinity+to+drive+unidirectional+growth+in+Arabidopsis.">Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.</a> Plant J. 66, 915-928 (2011).</li><li>Peng, F., 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=Fractional+separation+and+structural+features+of+hemicelluloses+from+sweet+sorghum+leaves.">Fractional separation and structural features of hemicelluloses from sweet sorghum leaves.</a> Bioresources. 7, (2012).</li><li>Xu, S. L., Rahman, A., Baskin, T. I., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+leucine-rich+repeat+receptor+kinases+mediate+signaling,+linking+cell+wall+biosynthesis+and+ACC+synthase+in+Arabidopsis.">Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis.</a> Plant Cell. 20, 3065-3079 (2008).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Imaging+of+Cellulose+Reorientation+during+Cell+Wall+Expansion+in+Arabidopsis+Roots.">Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots.</a> Plant Physiol. 152, 787-796 (2010).</li><li>Baskin, T. I., Beemster, G. T., Judy-March, J. E., Marga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorganization+of+cortical+microtubules+stimulates+tangential+expansion+and+reduces+the+uniformity+of+cellulose+microfibril+alignment+among+cells+in+the+root+of+Arabidopsis.">Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.</a> Plant Physiol. 135, 2279-2290 (2004).</li><li>Iyer, K. R. K., Neelakantan, P., Radhakrishnan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birefringence+of+native+cellulosic+fibers.+I.+Untreated+cotton+and+ramie.">Birefringence of native cellulosic fibers. I. Untreated cotton and ramie.</a> J. Polym. Sci. A-2: Polymer Physics. 6, 1747-1758 (1968).</li><li>Abraham, Y., Elbaum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+microfibril+angle+in+secondary+cell+walls+at+subcellular+resolution+by+means+of+polarized+light+microscopy.">Quantification of microfibril angle in secondary cell walls at subcellular resolution by means of polarized light microscopy.</a> New Phytol. 197, 1012-1019 (2013).</li><li>Petricka, J. J., Winter, C. M., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Arabidopsis+root+development.">Control of Arabidopsis root development.</a> Annu Rev Plant Biol. 63, 563-590 (2012).</li><li>Vanstraelen, M., Benkova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+Interactions+in+the+Regulation+of+Plant+Development.">Hormonal Interactions in the Regulation of Plant Development.</a> Annu Rev Cell Dev Biol. , (2012).</li><li>Singh, A. P., Savaldi-Goldstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control:+brassinosteroid+activity+gets+context.">Growth control: brassinosteroid activity gets context.</a> J Exp Bot. 66, 1123-1132 (2015).</li><li>Fridman, Y., 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=Root+growth+is+modulated+by+differential+hormonal+sensitivity+in+neighboring+cells.">Root growth is modulated by differential hormonal sensitivity in neighboring cells.</a> Genes Dev. 28, 912-920 (2014).</li><li>Vragovic, K., 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=Translatome+analyses+capture+of+opposing+tissue-specific+brassinosteroid+signals+orchestrating+root+meristem+differentiation.">Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.</a> Proc Natl Acad Sci U S A. 112, 923-928 (2015).</li><li>Hacham, Y., 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=Brassinosteroid+perception+in+the+epidermis+controls+root+meristem+size.">Brassinosteroid perception in the epidermis controls root meristem size.</a> Development. 138, 839-848 (2011).</li><li>Gu, Y., 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=Identification+of+a+cellulose+synthase-associated+protein+required+for+cellulose+biosynthesis.">Identification of a cellulose synthase-associated protein required for cellulose biosynthesis.</a> Proc Natl Acad Sci U S A. 107, 12866-12871 (2010).</li><li>Fendrych, M., 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=Programmed+cell+death+controlled+by+ANAC033/SOMBRERO+determines+root+cap+organ+size+in+Arabidopsis.">Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.</a> Curr Biol. 24, 931-940 (2014).</li></ol>

The authors have no conflicts of interest.

Here, we present a method for determination of the accumulation of crystalline cellulose in the different tissues composing the Arabidopsis roots, while maintaining anatomical information. As such, it provides an additional step towards understanding growth processes in plants at a cellular resolution. This method can be also applied for the study of additional plant species and organs.
A number of points must be considered when applying the method. First, crystalline cellulose accumulation in a given root section can be compared among tissues, among sections from different genotypes and among treatments if the orientation of their cellulose microfibrils is similar. Cellulose microfibrils orientation is determined in longitudinal section containing one face of the cell wall18. Hence, the series of longitudinal sections, covering the distinct tissues of the roots, from the outer to the inner most ones, enable analysis of the cellulose microfibrils orientation in the different tissues, at a given developmental stage. In the example shown here, the meristematic cells of the epidermis have a wide range of cellulose microfibrils orientations with an average cellulose microfibrils angle of 140&#176;, while the elongating cells of the epidermis have a more organized structure, with an average cellulose microfibrils angle of 120&#176;. These measurements were similar between wild type and the mutant of interest, thereby allowing a confident comparison of their relative crystalline cellulose accumulation, in the corresponding cell types and developmental zone.
Secondly, variability in the width of the different sections can impact the levels of light retardance measured. This is circumvented here by normalizing the raw retardance measurements of the tissue of interest (epidermal cells) to the retardance measurement of a different tissue (cortex cells). The normalized values are then compared (See Figure 2E-G).
Finally, transverse sections along the root, capture different developmental zones. Identification of these zones in the cross section is important for interpretation of the data. Loss of the outermost lateral root cap cell layer in the section comprise a straightforward zone marker. In general, cells start their fast expansion when the oldest cell of the lateral root cap undergoes programmed cell death, after which, the unprotected epidermis becomes the outermost tissue19.
Currently, methods that provide a cellular resolution measure of the ratio of the crystalline versus the amorphous cellulose domains are lacking. Indeed, this is also one limitation of the method presented here. However, its ability to communicate the accumulation of crystalline cellulose per se is a significant advance, as high relative levels of crystalline cellulose likely renders a more robust cell wall with increased tensile strength. Development of high resolution tools to precisely determine the composition the cell wall alongside its mechanical properties remains a future challenge.

The plant cell wall is a dynamic structure. Growing cells are surrounded by a primary cell wall, the organization of which allows cells to expand. Cells that cease to grow deposit a more rigid secondary wall that enhances the mechanical support of the plant. Both cell walls are composed of cellulose microfibrils embedded in a matrix of polysaccharides of different structures (e.g., hemicellulose and pectin) that vary across the different developmental stage and tissues1,2. Cellulose is synthesized as chains of (1,4)-&#946;-D-glucan that are tightly aligned to form the microfibril of a crystalline structure. Amorphous cellulose refers to the regions where the glucan chains are less ordered. The ratio between the crystalline and the amorphous domains is one parameter thought to affect the mechanical properties of the cell wall, by providing mechanical strength and viscoelastic characteristic, respectively3. Several methods have been developed to detect and quantify the two forms of cellulose arrangement, among them X-ray diffraction and cross polarization/magic angle spinning solid-state NMR4. X-ray diffraction can be used to determine the proportion of crystalline versus amorphous cellulose domains in the sample5. An alternative method uses fractionation of cell wall content into acid-insoluble and acid-soluble material, to distinguish between the crystalline and amorphous cellulose or other polymers, respectively. In this approach, incorporation of labeled glucose ([14C]Glucose) is used to quantify the cellulose6,7. These methods require large volumes of plant material for whole organ analyses, at best, and hence, are inadequately sensitive to tissue-specific variation in cell wall structure. Visualization of cellulose microfibrils at a cellular resolution can be achieved in live imaging studies combined with fluorescent dyes8,9, that can identify changes in the orientation of the cellulose microfibrils. However, these dyes are not used for quantification, they are not specific to crystalline cellulose and may interfere with the normal structure of the cell wall8. Polscope is an imaging technique that relies on the ability of crystalline cellulose to split light beams and retard part of the light10. Light retardation is strongest for microfibrils that lie perpendicularly to the direction of light propagation. For microfibrils with similar orientation, the higher the degree of crystallinity, the larger the light retardance11. Hence, polscope is used to study both the relative levels and orientation of the cellulose microfibrils.
Roots exhibit linear growth, during which cells originating at the stem cell niche, at the tip of the root, undergo a series of cell divisions, before they rapidly expand12. The cells comprising the root expand in a unidirectional (anisotropic) manner, as dictated by small molecule signaling hormones that impact the properties of the cell wall13. Differential responses to hormones, in time and space, provide a means of ensuring balanced organ growth14. Hence, high resolution analysis of cell wall structure can provide important information necessary to better understand the connection between cell type-specific responses to whole organ growth. Here, we report the implementation of polscope to study tissue-specific accumulation of crystalline cellulose in Arabidopsis roots, as observed in high quality anatomical sections. This method recently uncovered cell type-specific accumulation of crystalline cellulose in response to spatial perturbation of hormonal activity15.

<table>
	<tbody>
		<tr>
			<td>Murashige &#38; Skoog (MS)</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>LOBA CHEMIE&#160;</td>
			<td>6038</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Azure</td>
			<td>Sigma</td>
			<td>861065</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Driven 0.22mm PVDF Filter&#160;</td>
			<td>MILLEX-GV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>EMS</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma</td>
			<td>C-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica kit historesin&#160;</td>
			<td>Leica</td>
			<td>7022 18 500</td>
			<td></td>
		</tr>
		<tr>
			<td>embedding molds</td>
			<td>Agar Scientific</td>
			<td>AGG3530</td>
			<td></td>
		</tr>
		<tr>
			<td>film 100&#181; P.P.C.&#160;</td>
			<td>www.Jolybar.com</td>
			<td></td>
			<td>overhead film</td>
		</tr>
		<tr>
			<td>Knivemaker</td>
			<td>LKB</td>
			<td>7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultratome III</td>
			<td>LKB</td>
			<td>8800</td>
			<td>Ultratome</td>
		</tr>
		<tr>
			<td>Shandon immumount&#160;</td>
			<td>Thermo</td>
			<td>9990402</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio imaging system</td>
			<td>CRI</td>
			<td>Abrio imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio V2.2 software</td>
			<td>CRI</td>
			<td>Abrio V2.2 software</td>
			<td></td>
		</tr>
		<tr>
			<td>Open access polarized light image analysis software &#160;</td>
			<td>OPS</td>
			<td>OpenPolScope</td>
			<td><a href="http://www.openpolscope.org/">http://www.openpolscope.org/</a></td>
		</tr>
	</tbody>
</table>

high resolution quantification of crystalline cellulose accumulation in arabidopsis roots to monitor tissue-specific cell wall modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

We study the impact of cell type-specific responses to brassinosteroids (BRs), using the Arabidopsis root as a model organ15-17. When the BR receptor BRI1 is targeted, in the background of bri1 mutant, to a subset of epidermal cells called non-hair cells (Figure 2A,B), it inhibits unidirectional cell expansion of neighboring cells, and whole-root growth15. Polscope analysis was performed to reveal the mechanism underlying this inhibition. Longitudinal sections of the root obtained from wild type and from lines expressing BRI1 in non-hair cells only, showed similar orientation of cellulose microfibrils (Figure 2C,D,and as explained in 9.2.3), enabling comparison of the relative accumulation of crystalline cellulose in meristematic and elongating cells, using cross sections taken from these zones (Figure 2E,F). This analysis showed a correlation between BRI1 expression in non-hair cells and local accumulation of crystalline cellulose. Follow up experiments showed that mild inhibition of cellulose synthase by low concentrations of isoxaben lowered the level of crystalline cellulose in these cells, which in turn, partially restored growth inhibition, by promoting unidirectional cell expansion and root length15.
<img alt="Figure 1" src="/files/ftp_upload/53707/53707fig1.jpg" /> 
Figure 1: Polarized Light Microscopy System. The Polscope system consists of a light microscope (A), equipped with a CCD digital camera (B), analyzer and a green liquid crystal located on top of its light source (C). (D) Abrio software was used for image acquisition and analysis.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53707/53707fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53707/53707fig2.jpg" /> 
Figure 2: Polscope Analysis of Cell-Wall Composition. (A) Cross-section of the Arabidopsis primary root showing radial organization of its constituent tissues. Of these, the epidermal non-hair cells (N); hair cells (H) and cortex (C) are marked. Bar, 10 &#181;m. (B) Confocal microscopy images of roots expressing BRI1-GFP targeted to non-hair cells. White, PI staining that marks the cells, Green, BRI1-GFP expression. Bar, 20 &#181;m. (C) Longitudinal sections of roots obtained from wild type and from plants expressing BRI1 in non-hair cells. The angle of cellulose microfibrils (i.e., the angle between the color and cell long axis, reflecting the long axis of the root) is similar between the two lines. The cell wall flanking the cell along its long axis is encircled; angle mark represent the shaded cell wall which is measured. Bar, 50 &#181;m. (D) Quantification of cellulose microfibril angle in epidermal cell walls. Note the high variability of the angles present in meristematic cells (i.e., meristematic zone, MZ) as compared to cells in the transition zone (TZ), as reflected by the box plot. The similar average cellulose microfibril angle between wild type and plants expressing BRI1 in non-hair cells enables comparison of crystalline cellulose accumulation in cells of their corresponding developmental zone. The average angle in each sample is indicated by a red line. (E-F) Transverse root sections of these same plant backgrounds, shown in light retardance mode. Color scale represents light retardance of 0 - 17 nm. Sections corresponding to the meristem (E) and elongation (F) zones are shown. Bar, 50 &#181;m. The outer epidermal cell wall and inner cortical cell wall are encircled. (G) Quantification of the retardance values as calculated from the polscope images. Values are expressed as the ratio of retardance between the outer epidermal cell wall and the inner cortical cell wall. Note that the high deposition of crystalline cellulose in non-hair cells in lines expressing BRI1 in these cells only. Mean +SE; 40 &#60; n &#60; 600; (**) P &#60; 0.01; (***) P &#60; 0.001 in a two-tailed t-test. The figure was adopted and modified from15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53707/53707fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

high-resolution-quantification-crystalline-cellulose-accumulation

Nanyang Technological University

Research

JoVE Journal

Biology

8.1K Views.  Technion-Israel Institute of Technology. Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Video: High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

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Chapters in this video

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper