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://www.jove.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://www.jove.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://www.jove.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 ...

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Research

JoVE Journal

Biochemistry

3.6K Views.  University of Ottawa. Met dit protocol kunnen zaden intact worden gesneden, zodat ze niet afbrokkelen, waardoor zetmeelkorrels kunnen worden gevisualiseerd in elk zaad dat in een pipetpunt past. De kneedbare plastic kegel ondersteunt de inheemse structuur van de droge weefsels en organen, dus met dit goedkope en gemakkelijke spruitstuk kunnen veel monsters in korte tijd worden verwerkt. Om verder te gaan met het forensische microscopievoordeel hier, resistent zetmeel, iets waar we allemaal naar op zoek zijn, toega...

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

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

Neuroscience

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 ...

Biology

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

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 ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

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

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