Name: transverse sectioning of mature rice (oryza sativa l.) kernels for scanning electron microscopy imaging using pipette tips as immobilization support
Uploaded: 2022-01-25T13:00:00.000+00:00
Duration: 5 min 22 s
Description: Watch this Scientific Journal Video about Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support at JoVE.com

The authors are grateful to Systems for Research (SFR Corp.) for use of their Phenom ProX Desktop SEM instrument, as well as for the technical assistance provided by Maria Pilarinos (Systems for Research (SFR) Corp.) and Chlo&#235; van Oostende-Triplet (Cell Biology and Image Acquisition Core Facility, Faculty of Medicine, University of Ottawa). Funding was provided by the Low Carbon Innovation Fund (LCIF) from the Government of Ontario&#8217;s Ministry of Economic Development, Job Creation and Trade, and Proteins Easy Corp.

1. Preparation of transverse rice section
<ol>
	<li>De-husk dry, intact kernels as shown in Figure 1A.
	<ol>
		<li>Loosen the husks and dehull rice kernels by grinding the kernels between two flat rubber stoppers. Remove the husked rice kernels from the panicle, if necessary.</li>
		<li>Place a single kernel onto a flat rubber stopper on a work bench (Figure 1B). Ensure that this stopper remains stationary.</li>
		<li>Use a second flat rubber stopper (Figure 1C) to abrade the kernel by twisting it in against the first rubber stopper, using sufficient pressure (Figure 1D). Remove husks from the kernel, taking care not to shatter the endosperm. Remove any remaining husk using fine forceps. A de-husked kernel is shown in Figure 1E.</li>
	</ol>
	</li>
	<li>Using fine forceps, insert an individual husked kernel into a plastic pipette tip (250 &#181;L size, one seed/tip) (Figure 1F). Ensure that the embryo end of the kernel is facing towards the (conical) end of the pipette tip (Figure 1G). 
	NOTE: Inserting the kernel this way ensures that the kernel will fit as snugly as possible into the pipette tip as the kernel is narrower towards its proximal end.</li>
	<li>Insert a second 250 &#181;L pipette tip to force the kernel into the pipette tip and to keep the kernel immobile during sectioning, taking care not to damage the kernel or bend the second pipette tip (Figure 1H). The proper &#8216;telescope&#8217; assemblage is indicated in Figure 1I.</li>
	<li>Lay the pipette tip assemblage flat on a work bench and hold in place by hand (Figure 1J). With the other hand, use a sharp scalpel blade (No. 20) to slice through the center of the kernel and cut the end of the pipette tip off (Figure 1K). Using the scalpel cut 1 mm thick sections of the rice kernel (Figure 1L). 
	NOTE: The kernel section is tightly enclosed within an annulus of plastic (Figure 1M). Mean section thickness for three exemplar genotypes are found in Table 1. Sections significantly thinner than 1 mm will shatter or crumble. It is important to note from what part of the kernel each section originates if this experiment is being performed on several varieties of rice for comparison, as starch morphology does vary throughout the endosperm11.</li>
</ol>
2. Reflected light microscopy of transverse rice sections
<ol>
	<li>Use fine forceps to place the transverse rice sections (prepared in section 1) on a black piece of heavy gauge black paper.</li>
	<li>Obtain light images of transverse sections of Nipponbare using a stereomicroscope with mounted goosenecks for oblique illumination, as shown in Figure 1N-S.</li>
	<li>Observe endosperm morphology under at least 10x magnification. 
	NOTE: Any epilight source is preferable to bright field microscopy as sections obtained using this technique are not thin enough for the light to pass through.</li>
</ol>
3. Scanning electron microscopy of transverse rice sections
<ol>
	<li>Place the samples onto a carbon disk stuck to an aluminum stub and place onto a charge reduction sample holder. Remove the plastic ring&#160;from the pipette tip mount using fine forceps to prevent the plastic from entering the vacuum apparatus of the SEM. 
	NOTE: Images of endosperm cells, SGs, and subgranules are obtained using a desktop SEM machine that does not require samples to be sputter coated.</li>
	<li>Obtain the images using a high sensitivity multi-mode backscatter electron (BSE) detector at 10 kV.</li>
</ol>

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The authors have nothing to disclose.

The technique presented here represents a fast, simple, and keen approach towards preparing transverse rice cross sections for desktop SEM visualization. This sectioning technique allows for the rapid observation of endosperm structure, endosperm cell shape, size, and pattern, compound granules, and starch morphology. For the purposes of endosperm phenotyping and germplasm screening, it is critical to obtain an entire cross section of the rice kernel4,23,24. It is paramount to insert the kernel entirely within the pipette tip to prevent the pressure of the scalpel blade from forcing the endosperm to crumble or shatter. Provided the &#8216;telescope&#8217; assemblage is properly constructed, samples can be prepared for visualization within 15 seconds (Table 2) employing materials already in hand in a typical laboratory setting. This technique is applicable to the cross-sectioning of any ellipsoidal seed approximately four millimetres in diameter at its widest point. Seeds of the model grass Brachypodium distachyon (Figure S2A) can be similarly sectioned but do not stay enclosed within the annulus. Larger seeds, like wheat, fracture easily and require care when sectioning (Figure S2B).
However, there are several limitations to the technique presented here. Sections obtained using this technique are not thin enough for the light to pass through which prohibits the use of this technique for transmitted-light based microscopic approaches like bright field (500 &#181;m maximum sample thickness for rice kernel sections25) and transmission electron microscopy (TEM) (500 nm maximum sample thickness26). The use of a pipette tip as the sectioning &#8216;matrix&#8217; also limits the size of seed that can be sectioned using this technique. Further troubleshooting would be required to adapt this technique for species highly dissimilar from rice, and the size of the &#8216;matrix&#8217; is limited by the size of pipette tips available for purchase.
Another distinct advantage that this technique provides is the quality of samples that can be produced from chalky phenotype rice kernels. It is worth noting that even the Matsushima study conceded that it was difficult to obtain cross sections using that particular method for chalky phenotypes4, as replicated in this study for the purpose of comparison (Figure 1S). In their case, it became necessary to chemically fix their chalky rice samples and embed them in resin for sectioning. The new technique, in conjunction with desktop SEM imaging, allows the researcher to easily prepare transverse sections of rice kernels for microscopy with more consistency than without immobilization support (Table 3).
In the new era of phenomics and metabolomics, it is important to monitor mutagenized lines and transposon-tagged libraries to better understand the function and importance of starch in seeds. Additionally, the International Rice Genebank holds over 130 000 rice accessions27. A rapid seed phenotyping technique like the one presented here would expedite classification and sampling for nutritional quality28. Lastly, this technique may be useful in the light of encroaching climate change impacts. Seasonal high-temperature stress during grain-filling had already been identified as a major cause of chalkiness6, but recent studies have implicated rising global temperatures in increasing chalkiness of rice yields7,29. Such expedited endosperm phenotyping may help provide a broad agricultural image of the effect of increasing global temperatures.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family1,2. Endosperm phenotyping can be used to classify genotypes based on SG phenotype using scanning electron microscopic analysis. SGs can be visualized using scanning electron microscopy (SEM) by slicing the kernel and prying away the endosperm cell walls2.
The purpose of this technique is to easily prepare transverse rice kernel sections solely for the rapid SEM analysis. The development of this technique was motivated by the necessity for a rapid cross sectioning approach whereby samples are prepared for SEM microscopy immediately before visualization using minimal equipment.
This technique involves the insertion of the husked rice kernel into the pipette tip for complete immobilization. This is particularly important when cross-sectioning chalky rice kernel phenotypes, which are friable and easily crumble under pressure3. Chalkiness is an undesirable quality in rice since it affects the appearance of the kernel and causes the kernel to break easily&#160;during polishing and milling3. Chalkiness presents as an opaque area in a cross-section of the kernel that can be observed by the naked eye; at the microscopic level, chalkiness is characterized by small, loosely packed starch granules. Causes of chalkiness can be genetic4,5 or environmental6,7.
Cereal seed cross sections have traditionally been prepared using chemical fixing methods and sectioning following sample embedding into paraffin wax or another solid matrix4,8,9,10. In 2010, the Matsushima method was introduced as a way to avoid complicated and time-consuming rice kernel sample preparation4. This method involved the insertion of the husked rice kernel into a truncated pipette tip. The tip is held stationary by a block trimmer, and thin, partial endosperm sections are harvested using a hand-held razor blade. Another rapid technique developed in 2016 allowed for thin whole sectioning of a wide variety of dry seeds, including chalky varieties10. These methods motivated the development of the rapid technique presented here.
This new technique is appropriate for researchers who want to obtain intact transverse cross sections of rice kernels for endosperm phenotyping and starch morphology analysis using SEM.
This protocol represents an adaptation of the Matsushima truncated pipette tip method4, with several notable modifications: (1) kernels are not imbibed at any point of the technique; (2) neither a block trimmer nor an ultramicrotome are required to prepare the sections. A wild type &#8216;translucent&#8217; cultivar (Oryza sativa L. ssp. japonica cv. Nipponbare) and a mutagenized &#8216;chalky&#8217; line of Nipponbare (ssg1, substandard starch grain1)4 were examined in this study. These two cultivars were selected for the analysis here to demonstrate the technical and visual differences in processing translucent- and chalky-type rice sections.

<table><tbody><tr><td>JMP 15</td><td>SAS</td><td>N/A</td><td>N/A</td></tr><tr><td>Leit Adhesive Carbon Tabs 12 mm (Pack of 100)</td><td>Agar Scientific</td><td>AGG3347N</td><td>N/A</td></tr><tr><td>Phenom Pro Desktop SEM</td><td>Thermo Scientific</td><td>PHENOM-PRO</td><td>N/A</td></tr><tr><td>Pipette Tips RC UNV 250 &amp;micro;L</td><td>Rainin</td><td>17001116</td><td>N/A</td></tr><tr><td>SEM Pin Stub &amp;Oslash;12.7 Diameter Top, Standard Pin, Aluminium</td><td>Micro to Nano</td><td>10-002012-50</td><td>N/A</td></tr><tr><td>Shandon Microdissecting Fine Tips Thumb Forceps, Fine Tips, 12.7 cm</td><td>Thermo Scientific</td><td>3120019</td><td>N/A</td></tr><tr><td>Shandon Scalpel Blade No. 20, Sterile, 4.5 cm</td><td>Thermo Scientific</td><td>28618256</td><td>N/A</td></tr><tr><td>Shandon Stainless-Steel Scalpel Blade Handle</td><td>Thermo Scientific</td><td>5334</td><td>N/A</td></tr><tr><td>Zeiss V20 Discovery Stereomicroscope</td><td>Zeiss</td><td>N/A</td><td>N/A</td></tr></tbody></table>

transverse sectioning of mature rice (oryza sativa l.) kernels for scanning electron microscopy imaging using pipette tips as immobilization support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Wild type Nipponbare (Figure 2A) and ssg1 sections (Figure 2B) were examined under three magnifications: 260x, 920x, and 4200x. This technique allows for the preparation of sections of sufficient quality to observe the entire endosperm cell (Figure 3A), compound starch granules (Figure 3B), and individual subgranules (Figure 3C). Husked kernels take longer to process than polished kernels as the dry hulls need to be removed by abrasion before sectioning. Chalky kernels also take longer to process than polished translucent kernels, as care must be taken to not shatter the kernel during sectioning. A properly prepared rice section should be approximately 0.9 mm thick (Table 1) with minimal to no shattering of the endosperm (Figure 1N) and intact pericarp and aleurone layers (Figure 1O). Improper placement of the scalpel on the pipette tip when sectioning can lead to &#8216;chipped&#8217; sections (Figure 1P). Similarly, bright field images of optimal transverse sections of ssg1 (Figure 1Q) demonstrated intact endosperm, pericarp, and aleurone layers intact and available for visualization (Figure 1R). A broken chalky kernel section (Figure 1S) may still be useable for visualization if the only purpose is to observe SGs, but endosperm cell pattern will not be visible. A broken section may be difficult to handle for analysis. More shearing of endosperm cell walls was observed in wild type Nipponbare, as the cells are more tightly packed and less friable than the ssg1 kernels. No shearing of endosperm cells was observed in the ssg1 sections and compound starch granules are intact.
Figure S1 demonstrates the reliability of results using the &#8216;telescope&#8217; technique to section rice kernels. Rice lines identified as translucent kernel producers &#8211; wild type Resistant Starch (RS) hybrid line Xieyou 7954 (Oryza sativa L. ssp. indica)12,13,14 (Figure S1A) and cobalt-generated mutant RS11113,15 (Figure S1B) produced sections through which light was visible using a stereomicroscope. The corresponding SEM images revealed that these lines produce the &#8216;normal&#8217; rice endosperm phenotype: tightly packed, polyhedral starch granules. Chalky kernel producers, commercial variety Yi-Tang16 (Figure S1C) and RS413, a mutant of RS11115 (Figure S1D), exhibited white, opaque kernel sections. The corresponding SEM images displayed markedly different morphology compared to the wild type translucent RS background line: starch granules were round and loosely packed. Wild type Xiushui 11 (Oryza sativa L. ssp. japonica) (Figure S1E) and its mutant, KMD1 (Kemingdao1), which express the Cry1Ab gene to inhibit insect predation17,18,19 (Figure S1F) exhibited sections and endosperm morphotypes similar to the translucent RS lines.
The technique presented here is optimal for preparing samples of chalky-type rice kernels for phenotypic analysis, but also provides advantages for sectioning translucent rice kernel phenotypes20: slicing the samples using pressure from above reduces risk of shattering of the endosperm and dislocation. Samples can easily be prepared within seconds (Table 2). Multiple genotypes were analyzed using this technique to test its efficacy (Table 3). As shown in Figure S2, this technique can be applied to seeds of other species. The model monocot Brachypodium distachyon produces very hard seeds containing only B-granule starch21, which lack puroindoline A, a protein that confers softness to starch granules22. It was still possible to obtain an intact transverse section (Figure S2A). Obtaining an intact transverse section from soft white winter wheat (SWWW) was challenging but can be performed (Figure S2B). SWWW seeds are high in puroindoline A and large when compared to B. distachyon seeds and rice kernels. These seeds crumble frequently when sectioning using the telescope assemblage.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genotype</td>
			<td>Mean section width (&#181;m) using telescope assemblage</td>
			<td>Mean section width (&#181;m) sectioning freehand</td>
		</tr>
		<tr>
			<td>Nipponbare (husked)</td>
			<td>971.7 &#177; 152.4ab</td>
			<td>1059.571 &#177; 394.2ab</td>
		</tr>
		<tr>
			<td>Xieyou 7954</td>
			<td>825.1 &#177; 128.3b</td>
			<td>1306.187 &#177; 179.1a</td>
		</tr>
		<tr>
			<td>RS4</td>
			<td>910.6 &#177; 165.0ab</td>
			<td>1126.694 &#177; 395.3ab</td>
		</tr>
		<tr>
			<td colspan="3">Means followed by the same letters are not significantly different at P &#60; 0.01 using a one-way analysis of variance (ANOVA) and Tukey&#8217;s test (n = 10). Statistical analyses were performed using JMP 15 software.</td>
		</tr>
	</tbody>
</table>
Table 1: Mean kernel section thickness.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genotype</td>
			<td>Mean time (s)*</td>
		</tr>
		<tr>
			<td>Nipponbare (husked)</td>
			<td>14.7 &#177; 1.36a</td>
		</tr>
		<tr>
			<td>Xieyou 7954</td>
			<td>9.81 &#177; 0.98b</td>
		</tr>
		<tr>
			<td>RS4</td>
			<td>11.9 &#177; 1.28c</td>
		</tr>
		<tr>
			<td colspan="2">*Using the telescope assemblage.</td>
		</tr>
		<tr>
			<td colspan="2">Means followed by the same letters are not significantly different at P &#60; 0.01 using a one-way analysis of variance (ANOVA) and Tukey&#8217;s test (n = 10). Statistical analyses were performed using JMP 15 software.</td>
		</tr>
	</tbody>
</table>
Table 2: Mean sample preparation time.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Genotype</td>
			<td>Background</td>
			<td>Quality</td>
		</tr>
		<tr>
			<td>Nipponbare</td>
			<td>Wild type</td>
			<td>Translucent</td>
		</tr>
		<tr>
			<td>Substandard starch grain1 (ssg1)</td>
			<td>Nipponbare</td>
			<td>Chalky</td>
		</tr>
		<tr>
			<td>Resistant Starch (RS) Xieyou 7954</td>
			<td>Wild type</td>
			<td>Translucent</td>
		</tr>
		<tr>
			<td>RS111</td>
			<td>Xieyou 7954</td>
			<td>Translucent</td>
		</tr>
		<tr>
			<td>RS4</td>
			<td>RS111</td>
			<td>Chalky</td>
		</tr>
		<tr>
			<td>Yi-Tang, &#8216;New Life&#8217;, Lujuren brand</td>
			<td>Xieyou 7954</td>
			<td>Chalky</td>
		</tr>
		<tr>
			<td>Xiushui 11</td>
			<td>Wild type</td>
			<td>Translucent</td>
		</tr>
		<tr>
			<td>Kemingdao1 (KMD1)</td>
			<td>Xiushui 11</td>
			<td>Translucent</td>
		</tr>
	</tbody>
</table>
Table 3: Rice genotypes examined in this study.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61407/61407fig01.jpg" /> 
Figure 1: Preparation of transverse rice sections.&#160;(A) Wild type Nipponbare kernel with intact husk. (B). Kernel placed on a flat four-inch diameter rubber stopper. (C) Husks were removed by grinding the kernel between two apposing rubber stoppers. (D) Husk has been separated from the rice kernel. (E) Close-up of husked rice kernel. Embryo end is indicated. (F) Insertion of kernel into pipette tip using fine forceps. (G) Kernel was lodged into the distal end of the pipette tip. (H) Insertion of the second pipette tip to immobilize the kernel for sectioning (the &#8216;telescope&#8217; assemblage). (I) The rice kernel was fitted snugly into the distal end of the pipette tip. (J) Sectioning of the rice kernel within the assemblage. (K) Close-up of the section cut. (L) A section of the kernel enclosed by the plastic annulus. (M) Close-up of the transverse section. (N) Transverse section of wild type Nipponbare. (O) Close-up of the endosperm within the wild type Nipponbare section. (P) Poor, suboptimal section of wild type Nipponbare kernel. (Q) Transverse section of Nipponbare mutant ssg14. (R) Close-up of the endosperm within the ssg1 section. (S) Poor, suboptimal section of ssg1. Bar (panels A, N-S) = 1 mm. Whole rice kernel and sections were imaged using a stereomicroscope with a digital zoom camera and gooseneck lights. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61407/61407fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61407/61407fig02.jpg" /> 
Figure 2: SEM images of transverse kernel sections.&#160;(A) Wild type Nipponbare, a translucent cultivar. The compound starch granules were cemented tightly to one another; (B) Nipponbare mutant ssg14, a chalky phenotype. The compound starch granules were loosely packed and lack the cementitious nature of wild type Nipponbare starch morphotype. Magnification from left to right: 260x, 920x, and 4200x. Bar length is indicated in panels. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61407/61407fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61407/61407fig03.jpg" /> 
Figure 3: SEM microscopic anatomy of a transverse kernel section of Xiushui 11.&#160;(A) A single endosperm cell is outlined in red. 260x magnification. (B) A compound starch granule is outlined in red. 920x magnification. (C) Multiple starch subgranules are outlined in red. 2250x magnification. Bar lengths are indicated in the panels. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61407/61407fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure S1: Transverse sections of other rice genotypes prepared for SEM using this technique. (A) Resistant Starch (RS) Xieyou 795412. (B) RS111, a high-RS transparent mutant of 795413. (C) RS4, a chalky mutant of RS11115. (D) Yi-Tang, a commercial variety of high amylose rice16. (E) Xiushui 11. (F) KMD1 (Kemingdao1)17,18,19. 10x magnification for bright field images. White bar = 1 mm. 2250x magnification for SEM images. Bar lengths are indicated in the panels. <a href="https://cloudflare.jove.com/files/ftp_upload/61407/61407supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Figure S2: Technique is useful for other seeds. (A) Transverse section of false purple brome (Brachypodium distachyon L. accession Bd21) seed. (B) Transverse section of soft white winter wheat (Triticum aestivum L. cv. Augusta) seed. Bright field, 20x magnification. Bar = 1 mm. <a href="https://cloudflare.jove.com/files/ftp_upload/61407/61407supfig02.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support at JoVE.com

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

transverse-sectioning-mature-rice-oryza-sativa-l-kernels-for-scanning

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

3.6K Views.  University of Ottawa. This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Video: Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Engineering

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Developmental Biology

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.

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.

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 ...

Bioengineering

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Medicine

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.

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.

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 ...

Biology

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to observe plant tissues in three dimensions (3D) while preserving their internal structures. In rice (Oryza sativa L.), a monocot model plant and a globally important crop, clearing technology has been reported in organs that are relatively easy to observe, such as the roots and leaves. Applications of clearing technology in shoot apical meristem (SAM) and stems have also been reported, but only to a limited degree because of the poor penetration of the clearing solution (CS) in these tissues. The limited efficiency of the clearing solutions in these tissues has been attributed to auto-fluorescence, thickening, and hardening of the tissues in the stem as the vascular bundles and epidermis develop and layering of the SAM with water-repellent leaves. The present protocol reports the optimization of a clearing approach for continuous and 3D observation of gene expression from the SAM/young panicle to the base of the shoots during development. Fixed tissue samples expressing a fluorescent protein reporter were trimmed into sections using a vibrating micro-slicer. When an appropriate thickness was achieved, the CS was applied. By specifically targeting the central tissue, the penetration rate and uniformity of the CS increased, and the time required to make the tissue transparent decreased. Additionally, clearing of the trimmed sections enabled the observation of the internal structure of the whole shoot from a macro perspective. This method has potential applications in deep imaging of tissues of other plant species that are difficult to clear.

Deep Fluorescence Observation in Rice Shoots via Clearing Technology

The present protocol describes a clearing technique for rice shoots, which are difficult to prepare for internal structural observations owing to the hard, thick, or layered nature of the tissues. This method facilitates continuous and deep fluorescence observations, even in adult rice plants.

The recently developed clearing technology that eliminates refractive index mismatches and diminishes auto-fluorescent material has made it possible to ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Summary

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Chapters in this video

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

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

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

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

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

Deep Fluorescence Observation in Rice Shoots via Clearing Technology