Name: preparation and observation of thick biological samples by scanning transmission electron tomography
Uploaded: 2017-03-12T13:00:00
Description: Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

This work was funded by two ANR grants (ANR-11-BSV8-016 and ANR-10-IDEX-0001-02). We also acknowledge the PICT-IBiSA for providing access to chemical imaging equipment.

Precaución: Consulte las hojas de datos de seguridad del material (MSDS) de los diversos reactivos antes de usarlos. Varios de los productos químicos utilizados durante la preparación de la muestra son tóxicos, carcinogénicos, mutagénicos y / o tóxicos para la reproducción. Usar el equipo de protección personal (guantes, bata de laboratorio, pantalones largos y zapatos cerrados) y el trabajo bajo una campana de humos durante la manipulación de la muestra. Seccionar la muestra con un ultramicrotomo implica el u...

<ol>
	<li>De Rosier, D. J., Klug, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+three+dimensional+structures+from+electron+micrographs.">Reconstruction of three dimensional structures from electron micrographs.</a> Nature. 217 (5124), 130-134 (1968).</li><li>Gan, L., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+tomography+of+cells.">Electron tomography of cells.</a> Q Rev Biophys. 45 (1), 27-56 (2012).</li><li>Lucic, V., Rigort, A., Baumeister, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+tomography:+the+challenge+of+doing+structural+biology+in+situ.">Cryo-electron tomography: the challenge of doing structural biology in situ.</a> J Cell Biol. 202 (3), 407-419 (2013).</li><li>Al-Amoudi, A., 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=Cryo-electron+microscopy+of+vitreous+sections.">Cryo-electron microscopy of vitreous sections.</a> EMBO J. 23 (18), 3583-3588 (2004).</li><li>Koguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+STEM+for+observing+nanostructures.">Three-dimensional STEM for observing nanostructures.</a> J Electron Microsc (Tokyo). 50 (3), 235-241 (2001).</li><li>Midgley, P. A., Weyland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+electron+microscopy+in+the+physical+sciences:+the+development+of+Z-contrast+and+EFTEM+tomography.">3D electron microscopy in the physical sciences: the development of Z-contrast and EFTEM tomography.</a> Ultramicroscopy. 96 (3-4), 413-431 (2003).</li><li>Sousa, A. A., Azari, A. A., Zhang, G., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-axis+electron+tomography+of+biological+specimens:+Extending+the+limits+of+specimen+thickness+with+bright-field+STEM+imaging.">Dual-axis electron tomography of biological specimens: Extending the limits of specimen thickness with bright-field STEM imaging.</a> J Struct Biol. 174 (1), 107-114 (2011).</li><li>Sousa, A. A., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+STEM+for+the+biological+sciences.">Development and application of STEM for the biological sciences.</a> Ultramicroscopy. , 38-49 (2012).</li><li>Ercius, P., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incoherent+bright+field+STEM+for+imaging+and+tomography+of+ultra-thick+TEM+cross-sections.">Incoherent bright field STEM for imaging and tomography of ultra-thick TEM cross-sections.</a> Microsc Microanal. 15, (2009).</li><li>Hyun, J. K., Ercius, P., Muller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beam+spreading+and+spatial+resolution+in+thick+organic+specimens.">Beam spreading and spatial resolution in thick organic specimens.</a> Ultramicroscopy. 109 (1), 1-7 (2008).</li><li>Aoyama, K., Takagi, T., Hirase, A., Miyazawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STEM+tomography+for+thick+biological+specimens.">STEM tomography for thick biological specimens.</a> Ultramicroscopy. 109 (1), 70-80 (2008).</li><li>Biskupek, J., Leschner, J., Walther, P., Kaiser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+STEM+tomography+acquisition--a+comparison+of+convergent+beam+and+parallel+beam+STEM+tomography.">Optimization of STEM tomography acquisition--a comparison of convergent beam and parallel beam STEM tomography.</a> Ultramicroscopy. 110 (9), 1231-1237 (2010).</li><li>Hovden, 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=Breaking+the+Crowther+limit:+combining+depth-sectioning+and+tilt+tomography+for+high-resolution,+wide-field+3D+reconstructions.">Breaking the Crowther limit: combining depth-sectioning and tilt tomography for high-resolution, wide-field 3D reconstructions.</a> Ultramicroscopy. 140, 26-31 (2014).</li><li>Dahmen, T., 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=Combined+scanning+transmission+electron+microscopy+tilt-+and+focal+series.">Combined scanning transmission electron microscopy tilt- and focal series.</a> Microsc Microanal. 20 (2), 548-560 (2014).</li><li>Trepout, S., Messaoudi, C., Perrot, S., Bastin, P., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+transmission+electron+microscopy+through-focal+tilt-series+on+biological+specimens.">Scanning transmission electron microscopy through-focal tilt-series on biological specimens.</a> Micron. 77, 9-15 (2015).</li><li>Weyland, M., Muller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+convergence+angle+for+optimum+STEM+performance.">Tuning the convergence angle for optimum STEM performance.</a> FEI Nanosolutions. 1 (24), (2005).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7 (1), 27-41 (1998).</li><li>Forster, B., Van De Ville, D., Berent, J., Sage, D., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+wavelets+for+extended+depth-of-field:+a+new+method+for+the+fusion+of+multichannel+microscopy+images.">Complex wavelets for extended depth-of-field: a new method for the fusion of multichannel microscopy images.</a> Microsc Res Tech. 65 (1-2), 33-42 (2004).</li><li>Messaoudii, C., Boudier, T., Sanchez Sorzano, ., O, C., Marco, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TomoJ:+tomography+software+for+three-dimensional+reconstruction+in+transmission+electron+microscopy.">TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy.</a> BMC Bioinformatics. 8, 288 (2007).</li><li>Sorzano, C. O., 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=Marker-free+image+registration+of+electron+tomography+tilt-series.">Marker-free image registration of electron tomography tilt-series.</a> BMC Bioinformatics. 10, 124 (2009).</li><li>Kohl, L., Bastin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellum+of+trypanosomes.">The flagellum of trypanosomes.</a> Int Rev Cytol. 244, 227-285 (2005).</li><li>Luft, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+epoxy+resin+embedding+methods.">Improvements in epoxy resin embedding methods.</a> J Biophys Biochem Cytol. 9, 409-414 (1961).</li><li>Mielanczyk, L., Matysiak, N., Michalski, M., Buldak, R., Wojnicz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closer+to+the+native+state.+Critical+evaluation+of+cryo-techniques+for+Transmission+Electron+Microscopy:+preparation+of+biological+samples.">Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples.</a> Folia Histochem Cytobiol. 52 (1), 1-17 (2014).</li><li>Hovden, R., Xin, H. L., Muller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+depth+of+field+for+high-resolution+scanning+transmission+electron+microscopy.">Extended depth of field for high-resolution scanning transmission electron microscopy.</a> Microsc Microanal. 17 (1), 75-80 (2011).</li><li>Dahmen, T., 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=The+Ettention+software+package.">The Ettention software package.</a> Ultramicroscopy. 161, 110-118 (2016).</li><li>Murata, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+imaging+of+the+budding+yeast+Saccharomyces+cerevisiae+by+high-voltage+scanning+transmission+electron+tomography.">Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.</a> Ultramicroscopy. 146, 39-45 (2014).</li><li>Kizilyaprak, C., Daraspe, J., Humbel, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+scanning+electron+microscopy+in+biology.">Focused ion beam scanning electron microscopy in biology.</a> J Microsc. 254 (3), 109-114 (2014).</li><li>Kubota, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+electron+microscopy+for+serial+image+acquisition+of+neuronal+profiles.">New developments in electron microscopy for serial image acquisition of neuronal profiles.</a> Microscopy (Oxf). 64 (1), 27-36 (2015).</li><li>Levin, B. D., 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=Nanomaterial+datasets+to+advance+tomography+in+scanning+transmission+electron+microscopy.">Nanomaterial datasets to advance tomography in scanning transmission electron microscopy.</a> Sci Data. 3, 160041 (2016).</li></ol>

En este artículo, se presenta un protocolo de preparación de muestras convencional junto con una guía paso a paso para realizar el análisis 3D en muestras biológicas de espesor utilizando la tomografía por STEM-focal. Resina incrustación de muestras biológicas se ha utilizado durante décadas 26 y protocolos alternativos que se adaptan a diferentes tipos de muestras se pueden encontrar en la literatura 27. Por el contrario, las muestras gruesas de imagen en STEM es...

Desde principios de la década de 1970, la tomografía se acerca en la microscopía electrónica de transmisión (TEM) han sido ampliamente utilizados para la caracterización estructural de las muestras biológicas 1, 2, 3. El atractivo de la tomografía electrónica de transmisión era que podría ser utilizado para estudiar una amplia gama de estructuras biológicas a escala nanométrica, de la arquitectura de las células y la ultraestructura de los orgánulos a la estructura de los complejos macromoleculares y proteínas. Sin embargo, la tomografía electrónica de transmisión no se podría utilizar para estudiar muestras muy gruesas (mayor de 0,5 m). De hecho, las muestras gruesas producen demasiados electrones dispersos, la generación de imágenes de baja relación señal-ruido (SNR). Además, la tomografía implica la colección de imágenes de especímenes inclinados, con el espesor aparente de la muestra aumenta con el ángulo de inclinación. A pesar de que la dispersión inelástica se puede filtrarutilizando filtros de energía, la cantidad crítica de electrones necesarios para imágenes de alto SNR apenas se alcanza en TEM. Por lo tanto, las muestras biológicas de espesor sólo se han estudiado utilizando seccionamiento 4. Algunas muestras no pueden ser cortadas: algunos pueden degradar cuando se cortan, y otros necesitan ser estudiados en su totalidad con el fin de comprender su complejidad. Un enfoque alternativo es utilizar TEM en el modo 5, 6, 7, 8 de exploración. En microscopía de barrido electrónico de transmisión (STEM), la trayectoria óptica de los electrones es diferente de la de TEM convencional. Los electrones pasan a través de la muestra sin dispersión se pueden recoger en el eje óptico con un detector de campo brillante (BF) 9, mientras que dispersa elásticamente se pueden recoger en un cierto ángulo desde el eje óptico con un detector de campo oscuro (DF).La otra ventaja de STEM es que el haz de electrones enfocado es escaneado en la superficie de la muestra, lo que permite la colección de píxel por píxel de las imágenes. A pesar de que el haz de electrones se ensancha mientras pasa a través de la muestra 10, este sistema de recogida es menos sensible a los electrones inelástica dispersos que TEM convencional. Además, no hay lente post-espécimen en STEM, evitando así las aberraciones cromáticas que pueden ocurrir en TEM. La longitud de la cámara se puede ajustar de manera que el detector detecta BF principalmente electrones dispersados. Utilizando el detector DF para estudiar muestras gruesas, no se recomienda debido a la dispersión múltiple, que produce imágenes imprecisas. En lugar de ello, el detector de BF se puede utilizar 11. Mientras STEM puede producir imágenes de alta SNR, tiene una relativamente baja profundidad de campo debido a la relativamente alta convergencia de haz en comparación con TEM, disminuyendo la cantidad de información en profundidad que se puede recuperar a partir de espesorespecímenes. En el caso de microscopios de STEM de aberración corregida, en el que el ángulo de convergencia puede ser tan alta como 30 mrad, el campo de profundidad de puede ser lo suficientemente baja para que la información que está en el foco origina a partir de un plano focal de sólo unos pocos nanómetros . Configuración del haz de electrones en el modo paralelo aumenta la profundidad de campo del haz de electrones en detrimento de la resolución 12. Sin embargo, esta disposición no siempre es posible. Siempre que sea necesario el uso de un haz de electrones convergente, deberán utilizarse las técnicas que mejoran la profundidad de campo del haz de electrones en el estudio de muestras de espesor. Estudios recientes han informado de la adquisición de varias imágenes en diferentes planos focales en toda la muestra de recuperar la máxima cantidad de información que tiene el foco 13, 14. Los dos estudios describen diferentes maneras de manejar la información de los diferentes planos focales. Hovden et al.combinan en el espacio de Fourier de las imágenes que se recogieron en diferentes planos focales, y se obtuvo la reconstrucción final directamente a partir de la inversa 3D transformada de Fourier 13. Por el contrario, Dahmen et al. desarrollado un motor de reconstrucción de haz convergente para reconstruir en el espacio real del volumen 3D a partir de los diferentes planos focales 14. Nuestro laboratorio también desarrolló un método para obtener imágenes de muestras biológicas de espesor. Nuestra estrategia era diferente de los dos métodos descritos anteriormente en que nos fusionamos la información que está en el foco en los diferentes planos focales y reconstruido el volumen final en 3D en el espacio real mediante un proyector de haz paralelo 15. Nuestro objetivo fue desarrollar un método que se puede realizar fácilmente en cualquier laboratorio de microscopía electrónica. Con este fin, que tuvo como objetivo recoger las imágenes focales en una cantidad limitada de tiempo, comparable con el marco de tiempo de los experimentos de tomografía convencionales. Además, nuestra propuesta de método de could ser adaptado para su uso con diferentes tipos de alineación y software de reconstrucción. En el contexto de nuestra publicación a partir de 2015 15, queríamos visualizar y caracterizar la recuperación de la profundidad de campo, por lo que se utilizó una gran convergencia semi-ángulo de 25 mrad. A continuación, presentamos un protocolo paso a paso para realizar a través focal imágenes in STEM de acuerdo con el método desarrollado en nuestro laboratorio en 2015 15, y presentamos cómo se procesan los datos de 2015. Este método recupera la información que tiene el foco de varios planos focales a lo largo de un (750 nm) muestra biológica de espesor y permite reconstrucciones en 3D de alta calidad. En su caso, también se presentan las diferencias en esta metodología en comparación con los métodos utilizados por otros grupos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>EMS</td>
			<td>14120</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Add paraformaldehyde powder to PBS heated at approximately 60 &#176;C. 
			Increase pH by adding 1 N NaOH until no PFA powder is visible.</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxyde</td>
			<td>EMS</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73943</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Capsule</td>
			<td>EMS</td>
			<td>70110</td>
			<td></td>
		</tr>
		<tr>
			<td>Triming and Histo knives</td>
			<td>LFG Distribution</td>
			<td>Diatome diamond knives</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy copper grid</td>
			<td>LFG Distribution</td>
			<td>G200-Cu</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid Coating Pen</td>
			<td>LFG Distribution</td>
			<td>70624</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen Holder</td>
			<td>JEOL</td>
			<td>EM-21311 HTR</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscope</td>
			<td>JEOL</td>
			<td>JEM-2200FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and observation of thick biological samples by scanning transmission electron tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

En nuestro estudio, los electrones se aceleran a 200 kV en el microscopio electrónico de transmisión cañón de emisión de campo. Las imágenes se recogieron en modo STEM BF utilizando un tiempo de permanencia de 20 mu s. En cuanto al diseño de la serie de inclinación a través de-focal, encontramos que los intervalos de enfoque de 150 nm dieron resultados satisfactorios para el estudio ultraestructural de una muestra, incluso biológica 750 nm de espesor, aunque la profundidad de c...

Watch this Scientific Journal Video about Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography at JoVE.com

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Preparación y observación de muestras biológicas mediante el escaneo de grueso Electrónica de Transmisión de Positrones

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

The overall goal of this procedure is to obtain a three-dimensional reconstruction with depth of field recovery of a thick biological specimen using convergent beam scanning transmission electron tomography. This method can answer key questions in the field of structural biology about electron tomography of thick biological specimens. The main advantage of this technique is that images can be acquired in convergent bit mode and the data processing can be performed on most software dedicated to tomography.To begin preparing cell culture sample, centrifuge the sample at 5000 x g for five minutes. Use these centrifugation parameters throughout sample preparation. Discard the supernatant and resuspend the cells in one milliliter of PBS enriched with 4%paraformaldehyde and 2%gluteraldehyde.Incubate the suspension for 30 minutes at room temperature. Centrifuge the mixture, discard the supernatant, and wash the pellet three times with one milliliter portions of PBS. Using the same incubation conditions and washing procedure, incubate the pellet in one milliliter of PBS, enriched with 1%osmium tetroxide, and in one milliliter of PBS enriched with 2%uranyl acetate.Next, cool the sample to four degrees Celsius. Dehydrate the pellet by successive incubations in increasingly concentrated ethanol solutions. Maintaining the sample temperature of four degrees Celsius throughout incubation, centrifugation, and washing.Resuspend the dehydrated pellet in one milliliter of pure ethanol, and incubate overnight at 4 degrees Celsius. Then, allow the sample to warm to room temperature, and centrifuge the sample. Incubate the pellet in increasingly concentrated epoxy resin solutions at room temperature.After the final wash, resuspend the pellet in one milliliter of pure epoxy resin, and incubate overnight at room temperature. Centrifuge the sample, and remove the supernatant, and then resuspend the pellet in 200 microliters of epoxy resin and hardener. Transfer the suspension into the capsule.Incubate the plastic capsule at 60 degrees Celsius for 48 hours to polymerize the resin. Remove the capsule from the incubator and verify resin polymerization. Add a layer of epoxy resin and hardener over the embedded sample and incubate the capsule again under the same conditions.Mount the sample embedded in resin upside down in a specimen holder. Securely fix the sample on the trimming block of an ultramicrotome. Cut the plastic capsule away from the sample in the resin.Shape the exposed resin into a pyramid. Then transfer the capsule and specimen holder to the moving arm of the ultramicrotome. Install a trimming knife and guided by a microscope, refine the pyramid shape.Replace the trimming knife with a histology knife. Under the microscope, adjust the capsule pitch angle so the pyramid is perpendicular to the knife edge. Set the cutting speed and section the sample.After sectioning the sample, transfer the chosen section to a treated copper grid over filter paper. Allow the sample to dry, and then load the sample into the electron microscope. To design the through focal tilt series, first calculate the electron beam depth of field and the maximal apparent thickness of the sample.Based on these values, select a focus interval and determine the number of focal steps needed to image the entire sample at the maximal apparent thickness. Then, at low magnification, locate the region of interest within the sample. Adjust the eucentric height so that the ROI does not appear to move when the sample is rotated.Verify that the ROI remains visible throughout the tilt range. Fill in the through focal tilt series parameters for automated image collection and acquire the images. Import the image series into an image processing program, and align the images collected at the same tilt angle in a pyramidal hierarchy.Find the alignment at each tilt angle by stacking the aligned images. Browse through the stack to ensure that only the focused region moves from image to image and realign the images as needed. Once alignment is verified throughout the sample, merge the in-focus information at the high settings to yield the series with a single image per tilt angle.Perform find alignment of the tilt series based on local minima or fiducial markers. Perform a 3-D reconstruction and inspect the resulting image. If the reconstruction appears blurred or deformed, realign the original image series.A sample of t. brucei was imaged and analyzed using this method. At a zero degree tilt angle, several details of the flagellar pocket are visible.At the higher tilt angle of 70 degrees, only a portion of each image collected in the through focal series is in focus. This position shifts throughout the series. By merging the in-focus sections, a single image with a larger in-focus zone is produced.This effect is even more pronounced when the high frequency areas of the through focal tilt series are highlighted. The high frequency information spans most of the combined image, indicating that the through focal tilt series has been processed well. Once mastered, the image processing part can be done in around three to four hours, depending on the chosen alignment and reconstruction parameters.While attempting this procedure, it's important to remember that the quality of the final reconstruction greatly depends on the quality of the previous alignments tests. After watching this video, you should have a good understanding of how to perform the collection and the image processing of a through focal tilt series.

Research

JoVE Journal

Biology

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

La visualización de la ATP sintasa dímeros en mitocondrias por Electron Cryo-tomografía

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Investigación

Biología

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje<em&gt; In situ</em&gt; La hibridación

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

El estudio de la Organización Supramolecular de membranas fotosintéticas dentro de los tejidos foliares fracturados-Freeze por Cryo-Microscopía Electrónica de Barrido

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Escaneado de Protocolos de Microscopía Electrónica (SEM) para la Problemática de la planta, Oomycete, y muestras de hongos

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Preparación de la muestra y proyección de imagen de exosomas por microscopía electrónica de transmisión

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

Visualización de compactación del ADN en cianobacterias por Tomography del Cryo-electrón de alta tensión

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Preparación de muestras miniatura para microscopia electrónica de transmisión

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Preparación de muestras biológicas por congelación de alta presión, realce del contraste asistida por microondas y mínima inclusión de proyección de imagen de volumen

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

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

Flujo de trabajo de tomografía de matriz para la adquisición dirigida de información de volumen mediante microscopía electrónica de barrido

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

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

Microscopía Electrónica De Barrido Bloque-Cara Serial (SBF-SEM) De Muestras De Tejido Biológico

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...