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>

<ol>
	<li>James, M. G., Denyer, K., Myers, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+synthesis+in+the+cereal+endosperm.">Starch synthesis in the cereal endosperm.</a> Current Opinion in Plant Biology. 6 (3), 215-222 (2003).</li><li>Shapter, F. M., Henry, R. J., Lee, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosperm+and+starch+granule+morphology+in+wild+cereal+relatives.">Endosperm and starch granule morphology in wild cereal relatives.</a> Plant Genetic Resources. 6 (2), 85-97 (2008).</li><li>Ashida, K., Iida, S., Yasui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological,+physical,+and+chemical+properties+of+grain+and+flour+from+chalky+rice+mutants.">Morphological, physical, and chemical properties of grain and flour from chalky rice mutants.</a> Cereal Chemistry. 86 (2), 225-231 (2009).</li><li>Matsushima, R., Maekawa, M., Fujita, N., Sakamoto, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+direct+observation+method+to+isolate+mutants+with+defects+in+starch+grain+morphology+in+rice.">A rapid, direct observation method to isolate mutants with defects in starch grain morphology in rice.</a> Plant and Cell Physiology. 51 (5), 728-741 (2010).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+stable+QTLs+causing+chalk+in+rice+grains+in+nine+environments.">Identification of stable QTLs causing chalk in rice grains in nine environments.</a> Theoretical and Applied Genetics. 129 (1), 141-153 (2016).</li><li>Nagato, K., Ebata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high+temperature+during+ripening+period+on+the+development+and+the+quality+of+rice+kernels.">Effects of high temperature during ripening period on the development and the quality of rice kernels.</a> Japanese Journal of Crop Science. 34 (1), 59-66 (1965).</li><li>Zhao, X., Fitzgerald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Climate+change:+implications+for+the+yield+of+edible+rice.">Climate change: implications for the yield of edible rice.</a> PLoS One. 8 (6), 66218 (2013).</li><li>Zhao, Z. K., Mu, T. H., Zhang, M., Richel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high+hydrostatic+pressure+and+microbial+transglutaminase+treatment+on+structure+and+gelation+properties+of+sweet+potato+protein.">Effects of high hydrostatic pressure and microbial transglutaminase treatment on structure and gelation properties of sweet potato protein.</a> LWT - Food Science and Technology. 115, 108436 (2019).</li><li>Feiz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Puroindolines+co-localize+to+the+starch+granule+surface+and+increase+seed+bound+polar+lipid+content.">Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content.</a> Journal of Cereal Science. 50 (1), 91-98 (2009).</li><li>Zhao, L., Pan, T., Guo, D., Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+preparing+the+whole+section+of+starchy+seed+to+investigate+the+morphology+and+distribution+of+starch+in+different+regions+of+seed.">A simple and rapid method for preparing the whole section of starchy seed to investigate the morphology and distribution of starch in different regions of seed.</a> Plant Methods. 14 (1), 16 (2018).</li><li>Zhao, L., Pan, T., Cai, C., Wang, J., Wei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+whole+sections+of+mature+cereal+seeds+to+visualize+the+morphology+of+endosperm+cell+and+starch+and+the+distribution+of+storage+protein.">Application of whole sections of mature cereal seeds to visualize the morphology of endosperm cell and starch and the distribution of storage protein.</a> Journal of Cereal Science. 71, 19-27 (2016).</li><li>Li, C., Dong, S., Li, G., Yuan, G., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+and+application+of+the+new+combination+of+hybrid+rice+"Xieyou+7954".">Breeding and application of the new combination of hybrid rice "Xieyou 7954".</a> Journal of Zhejiang University (Agriculture and Life Sciences). 19 (3), 179-181 (2002).</li><li>Shu, X., Jia, L., Ye, H., Li, C., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+digestion+properties+of+rice+different+in+resistant+starch.">Slow digestion properties of rice different in resistant starch.</a> Journal of Agricultural and Food Chemistry. 57 (16), 7552-7559 (2009).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+roles+of+soluble+starch+synthase+SSIIIa+and+granule-bound+starch+synthase+Waxy+in+synthesizing+resistant+starch+in+rice.">Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice.</a> Proceedings of the National Academy of Sciences. 113 (45), 12844-12849 (2016).</li><li>Yang, C. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+properties+of+mutant+rice+high+in+resistant+starch.">Starch properties of mutant rice high in resistant starch.</a> Journal of Agricultural and Food Chemistry. 54, 523-528 (2006).</li><li>Zhou, Y., Zou, Y., Jiang, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+methods+for+resistance+starch+content+of+Yi-Tang+rice+and+optimization+of+pretreatment.">Detection methods for resistance starch content of Yi-Tang rice and optimization of pretreatment.</a> Food Science and Biotechnology. 36, 416-419 (2017).</li><li>Cheng, X., Sardana, R., Kaplan, H., Altosaar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-transformed+rice+plants+expressing+synthetic+cryIA(b)+and+cryIA(c)+genes+are+highly+toxic+to+striped+stem+borer+and+yellow+stem+borer.">Agrobacterium-transformed rice plants expressing synthetic cryIA(b) and cryIA(c) genes are highly toxic to striped stem borer and yellow stem borer.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (6), 2767-2772 (1998).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+P-35S+and+T-nos+in+genetically+modified+organisms+by+recombinase+polymerase+amplification+combined+with+a+lateral+flow+strip.">Rapid detection of P-35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip.</a> Food Control. 107, 106775 (2020).</li><li>Shu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rice+plants+with+a+synthetic+cry1Ab+gene+from+Bacillus+thuringiensis+were+highly+resistant+to+eight+lepidopteran+rice+pest+species.">Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species.</a> Molecular Breeding. 6 (4), 433-439 (2000).</li><li>Lisle, A. J., Martin, M., Fitzgerald, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chalky+and+translucent+rice+grains+differ+in+starch+composition+and+structure+and+cooking+properties.">Chalky and translucent rice grains differ in starch composition and structure and cooking properties.</a> Cereal Chemistry. 77 (5), 627-632 (2000).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+development+of+starch+granules+and+the+regulation+of+starch+biosynthesis+in+Brachypodium+distachyon:+comparison+with+common+wheat+and+Aegilops+peregrina.">Dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon: comparison with common wheat and Aegilops peregrina.</a> BMC Plant Biology. 14 (1), 198 (2014).</li><li>Giroux, M. J., Morris, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheat+grain+hardness+results+from+highly+conserved+mutations+in+the+friabilin+components+puroindoline+a+and+b.">Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (11), 6262-6266 (1998).</li><li>Matsushima, R., Hisano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+amyloplasts+in+the+developing+endosperm+of+barley+and+rice.">Imaging amyloplasts in the developing endosperm of barley and rice.</a> Scientific Reports. 9, 3745 (2019).</li><li>Matsushima, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloplast-localized+SUBSTANDARD+STARCH+GRAIN4+protein+influences+the+size+of+starch+grains+in+rice+endosperm.">Amyloplast-localized SUBSTANDARD STARCH GRAIN4 protein influences the size of starch grains in rice endosperm.</a> Plant Physiology. 164 (2), 623-636 (2014).</li><li>Monjardino, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+flange+and+reticulate+wall+ingrowths+in+maize+(Zea+mays+L.)+endosperm+transfer+cells.">Development of flange and reticulate wall ingrowths in maize (Zea mays L.) endosperm transfer cells.</a> Protoplasma. 250 (2), 495-503 (2013).</li><li>Tizro, P., Choi, C., Khanlou, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+for+transmission+electron+microscopy.">Sample preparation for transmission electron microscopy.</a> Methods in Molecular Biology. 1897, 417-424 (2019).</li><li>Liu, Q. H., Zhou, X. B., Yang, L. Q., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chalkiness+on+cooking,+eating+and+nutritional+qualities+of+rice+in+two+indica+varieties.">Effects of chalkiness on cooking, eating and nutritional qualities of rice in two indica varieties.</a> Rice Science. 16 (2), 161-164 (2009).</li><li>Morita, S., Wada, H., Matsue, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Countermeasures+for+heat+damage+in+rice+grain+quality+under+climate+change.">Countermeasures for heat damage in rice grain quality under climate change.</a> Plant Production Science. 19 (1), 1-11 (2016).</li></ol>

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 &#181;L</td>
			<td>Rainin</td>
			<td>17001116</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>SEM Pin Stub &#216;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 ...

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

Research

JoVE Journal

Biochemistry

3.5K 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

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...