This research was supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT (19-BR-01-08), and National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)(No. 2019R1A2C1010634). SCO1-APEX2 and HRP-KDEL plasmids were kindly provided by Hyun-Woo Rhee (Seoul National University). TEM data were acquired at Brain Research Core Facilities in KBRI.

1. Cell culture with patterned grid culture dish and cell transfection with SCO1-APEX2 and HRP-KDEL plasmid vector
<ol>
	<li>Seed 1 x 105 HEK293T cells by placing them into 35-mm glass grid-bottomed culture dishes in a humidified atmosphere containing 5% CO2 at 37 &#176;C in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.</li>
	<li>The day after seeding the cells, when they have grown to 50%-60% conflue...

<ol>
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Determining the cellular localization of specific proteins at a nanometer resolution using EM is crucial to understand the cellular functions of proteins. Generally, there are two techniques to study the localization of a target protein via EM. One is the immunogold technique, which has been used in EM since 1960, and the other is a technique using recently developed genetically encoded tags16. Traditional immunogold techniques have employed antibody-conjugated gold particles or quantum dots to sh...

Mitochondria and endoplasmic reticulum (ER) are membrane-bound cellular organelles. Their connection is necessary for their function, and proteins related to their network have been described1. The distance between the mitochondria and ER has been reported as approximately 100 nm using light microscopy2; however, recent super-resolution microscopy3 and electron microscopy (EM)4 studies have revealed it to be considerably smaller, at approximately 10-25 nm. The resolution achieved in super-resolution microscopy is lower than EM, and specific labeling is necessary. EM is a suitable technique to attain a sufficiently high-resolution contrast for structural studies of the connections between mitochondria and ER. However, a disadvantage is the limited z-axis information because the thin sections must be approximately 60 nm or thinner for conventional transmission electron microscopy (TEM). For sufficient EM z-axis imaging, three-dimensional electron microscopy (3DEM) can be used5. However, this involves the preparation of hundreds of thin serial sections of whole cells, which is very tricky work that only a few skilled technologists have mastered. These thin sections are collected on fragile formvar film-coated one-hole TEM grids. If the film breaks on one gird, serial imaging and volume reconstruction is not possible. Serial block-face scanning electron microscopy (SBEM) is a popular technique for 3DEM that uses destructive en bloc sectioning inside the scanning electron microscope (SEM) vacuum chamber with either a diamond knife (Dik-SBEM) or a focused ion beam (FIB-SEM)6. However, because those techniques are not available at all facilities, we suggest array tomography7 using serial sectioning and SEM. In array tomography, serial sections cut using an ultramicrotome are transferred to a glass coverslip instead of a TEM grid and visualized via light microscopy and SEM8. To enhance the signal for backscatter electron (BSE) imaging, we utilized an en bloc EM staining protocol employing osmium tetroxide (OsO4)-fixed cells with osmiophilic thiocarbohydrazide (TCH)9, enabling us to obtain images without post-embedding double staining.
Additionally, the mitochondrial marker SCO1 (cytochrome c oxidase assembly protein 1)&#8211; ascorbate peroxidase 2 (APEX2)10 molecular tag was used to visualize mitochondria at the EM level. APEX2 is approximately 28 kDa and is derived from soybean ascorbate peroxidase11. It was developed to show the detailed location of specific proteins at the EM level in the same way that green fluorescent protein-tagged protein is used in light microscopy. APEX2 converts 3,3&#39;&#160;-diaminobenzidine (DAB) into an insoluble osmiophilic polymer at the site of the tag in the presence of the cofactor hydrogen peroxide (H2O2). APEX2 can be used as an alternative to traditional antibody labeling in EM, with a protein localization throughout the depth of the entire cell. In other words, the APEX2-tagged protein can be visualized by specific osmication11 without immunogold labeling and permeabilization after ultra-cryosectioning. Horseradish peroxidase (HRP) is also a sensitive tag that catalyzes the H2O2-dependent polymerization of DAB into a localized precipitate, providing EM contrast after treatment with OsO4. The ER target peptide sequence HRP-KDEL (lys-asp-glu-leu)12 was applied to visualize ER within a whole cell. To evaluate our protocol of utilizing genetic tags and en bloc staining with reduced osmium and TCH (rOTO method), using the osmication effect at the same time, we compared the membrane contrast with and without the use of each genetic tag in rOTO en bloc staining. Although 3DEM with array tomography and DAB staining with APEX and HRP have, respectively, been utilized for other purposes, our protocol is unique because we have combined array tomography for 3DEM and DAB staining for mitochondria and ER labeling. Specifically, we showed five cells with and without APEX-tagged genes in the same section, which aided in investigating the effect of the genetic modification on cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:869px;" width="868">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glutaraldehyde</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">16200</td>
			<td style="width:349px;">Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Paraformaldehyde</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">19210</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Sodium cacodylate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">12300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Osmium tetroxide 4 % aqueous solution</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">16320</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Epon 812</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">14120</td>
			<td>EMbed 812- 20 ml/ DDSA- 16 ml/ NMA- 8 ml/ DMP-30 - 0.8 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Ultra-microtome Leica ARTOS 3D</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">ARTOS 3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Uranyl acetate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">22400</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lead citrate</td>
			<td style="width:123px;">EMS</td>
			<td style="width:123px;">17900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">35mm Gridded coverslip dish</td>
			<td style="width:123px;">Mattek</td>
			<td style="width:123px;">P35G-1.5-14-CGRD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glow discharger</td>
			<td style="width:123px;">Pelco</td>
			<td style="width:123px;">easiGlow</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Formvar carbon coated Copper Grid</td>
			<td style="width:123px;">Ted Pella</td>
			<td style="width:123px;">01805-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hydrochloric acid</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">258148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fugene HD</td>
			<td style="width:123px;">Promega</td>
			<td style="width:123px;">E2311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Glycine</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">G8898</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:275px;">3,3&#8242; -diaminobenzamidine (DAB)</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">D8001</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Hydrogen peroxide solution</td>
			<td style="width:123px;">Merck</td>
			<td style="width:123px;">107210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate(II) trihydrate</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">P3289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">0.22 um syringe filter</td>
			<td style="width:123px;">Sartorius</td>
			<td style="width:123px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiocarbonyldihydrazide</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">223220</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Potassium hydroxide</td>
			<td style="width:123px;">Fluka</td>
			<td style="width:123px;">10193426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">L-aspartic acid</td>
			<td style="width:123px;">SIGMA</td>
			<td style="width:123px;">A9256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Ethanol</td>
			<td style="width:123px;">Merck</td>
			<td style="width:123px;">100983</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Transmission electron microscopy</td>
			<td style="width:123px;">FEI</td>
			<td style="width:123px;">Tecnai G2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Indium tin oxide (ITO) coated glass coverslips</td>
			<td style="width:123px;">SPI</td>
			<td style="width:123px;">06489-AB</td>
			<td>fragil glass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Isopropanol</td>
			<td style="width:123px;">Fisher Bioreagents</td>
			<td style="width:123px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Diamond knife</td>
			<td style="width:123px;">Leica</td>
			<td style="width:123px;">AT-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Inveted light microscopy</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:123px;">ECLipse TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Scanning electron microscopy</td>
			<td style="width:123px;">Zeiss</td>
			<td style="width:123px;">Auriga</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondria and endoplasmic reticulum imaging by correlative light and volume electron microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

Figure 1 describes the schedule and workflow for this protocol. The protocol requires 7 days; however, depending on the time spent on SEM imaging, this may increase. For cell transfection, the confluency of the cells should be controlled so as not to cover the bottom of the entire grid plate (Figure 1A). A high cell density could prevent the identification of the cell of interest during light microscope and EM observation. We used genetically tagged plasmids that expressed APEX2 and HRP ...

Watch this Scientific Journal Video about Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy at JoVE.com

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

This volume electron microscopy technique is used to perform biological studies that require the observation of a 3-D structure. Offering a rod, fielder view, and sample storage before image retakes. Fx2 Asan engineered the path stages that catalyzes the deabilation to increase the electron test of target structures and can be used to locate a specific target of interest.These particles combines the use of correlative light and 3-D electron microscopy technique to investigate the interactions between membranous organelles and host cells. 16 to 24 hours after transfection, gently wash the culture with 250 microliters of 30 to 37 degree Celsius fixation solution. Quickly replacing the wash with 1.5 milliliters of fresh fixation solution, and placing the culture on ice for 30 minutes.At the end of the incubation, rinse the cells with three ten minute washes in one milliliter of ice-cold 0.1 molar sodium cacodylate buffer per wash. After the last wash, add one milliliter of ice-cold 0 to 4 degrees Celsius 20 millimolar glycine solution to the dish for a ten-minute incubation on ice followed by three five minute washes with one milliliter of fresh, cold 0.1 molar sodium cacodylate buffer per wash. Next, label the cells with 500 microliters of freshly prepared DAB solution.And incubate the cells on ice for approximately five to forty-five minutes. When a light brown stain is visible under an inverted light microscope, rinse the cells three times with one milliliter of fresh, cold 0.1 molar sodium cacodylate buffer for ten minutes per wash. Then, use a phase contrast inverted microscope to visualize the DAB staining at a 100 times magnification or higher and mark the bottom of the glass where the region of interest is located.For electron microscope block sample preparation, first post-fix the cells with one milliliter of 2%reduced osmium tetroxide for one hour at four degrees Celsius. At the end of the fixation, rinse the cells with three five-minute washes and one milliliter of fresh distilled water per wash. Before treating the cells with one milliliter of filtered TCH solution for 20 minutes.At the end of the incubation, wash the cells three times in distilled water as just demonstrated and fix the cells with a 30-minute incubation in 2%reduced osmium tetroxide. At the end of the second fixation, wash the cells three times with distilled water and cover the culture with one milliliter of aqueous 1%uranyl acetate overnight at 4 degrees Celsius protected from light. The next morning, wash the cells three times with distilled water before staining with one milliliter of 60 degree Celsius warmed Walton's lead aspartate solution for 30 minutes in a 60 degrees Celsius oven.At the end of the incubation, wash the cells three times in distilled water. And incubate the culture in a graded series of 20-minute 2 milliliter ethanol aliquots. After the last 100%ethanol exposure, incubate the cells for 30 minutes in one milliliter of three to one ethanol to low viscosity embedding mixture medium at room temperature.At the end of the incubation, replace the medium with one milliliter of one-to-one ethanol to low viscosity embedding mixture medium for another 30 minute incubation at room temperature. Next, replace the medium with one millilter of one-to-three ethanol to low viscosity embedding mixture medium for an additional 30 minutes at room temperature. Then, replace the medium with one milliliter of 100%low viscosity embedding medium overnight, followed by embedding of the sample in 100%low viscosity embedding mixture for 24 hours at 60 degrees Celsius.For transmission electron microscopy analysis, the next day, use an ultra microtome to obtain 90 nanometer thick sections and observe the grid under transmission electron microscopy at 200 kilovolts. Before beginning the sample preparation, clean Indium Tin Oxide coated glass coverslips with gentle agitation in isopropanol for 30 to 60 seconds. At the end of the agitation, drain off the excess alcohol and place the coverslips in a dust-free environment.When the coverslips are dry, use a plasma coater to glow discharge the coverslips for one minute. Then insert the coverslips into the substrate holder and place the substrate holder into the knife boat. To obtain samples for scanning electron microscopy, insert the embedded sample block into the sample holder of the ultramicrotome.And set the holder into the trimming block. Use a razor blade to trim away the excess resin around the target position so that the face of the block acquires a trapezoid or rectangular shape. The leading and trailing edges should be strictly parallel to each other.It is not easy to acquire a large number of serial sections. However, you have to have the leading edges and trailing edges completely parallel. Next, insert the sample holder into the arm of the ultramicrotome.Place the diamond knife in the knife holder, and fill the knife boat with filtered distilled water. Then, carefully push the handle of the holder to adjust the carrier position so that the edge of the coverslips is close to the knife. After obtaining the sample, stop the sectioning process, slowly open the clamping screw of the tube and drain the water boat.When the ribbon collection process is complete, remove the substrate with the handle of the clamping device and dry the ribbon. The use of genetically tagged plasmids expressing proteins of interest, allows the identification of the transfected target cells after staining for the tagged proteins. Correlative light electron microscopy can then be used to further visualize the labeled cell culture.For example, the dark stain generated by SCO1-APEX2 is observed exclusively in the mitochondrial intermembrane space and not in the matrix space of the mitochondria. Code transfected cells with both SCO1-APEX2 and HRP-KDEL plasmids express a highly dense electron signal in the mitochondrial intermembrane space and the endoplasmic reticulum. However, no endoplasmic reticulum staining is observed in cells that are transfected with SCO1-APEX2 only.For serial imaging by scanning electron microscopy, an overview of the whole array image is created using a backscatter electron detector over a large area. Next, the region of interest is placed in the first section and propagated to all the other sections. Finally, the region of interest containing five target cells is imaged with five nanometer imaged pixels.Magnification reveals detailed subcellular structures such as the Golgi apparatus, mitochondria, and endpolasmic reticulum. Serial imaging clearly reveals that endoplasmic reticulum-mitochondria contacts occur on different z-planes. You should have a good understanding of how to locate a specific target structure using correlative light and electron microscopy to investigate the effects of a specific identity modification.These staining techniques combined with volume electron microscopy could be used for larger scale EM to investigate cellular interactions particularly in eurovirus. Remember the glutaraldehyde, also enteterocide and TCH are very toxic, so be sure to wear protective clothing and to work in a fume hood when using these materials.

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Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

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12.6K vistas.  Korea Brain Research Institute. Esta técnica de microscopía electrónica de volumen se utiliza para realizar estudios biológicos que requieren la observación de una estructura 3D. Ofreciendo una varilla, una vista de jardinero y un almacenamiento de muestras antes de que se vuelva a tomar la imagen. Fx2 Asan diseñó las etapas de ruta que catalizan la deabilización para aumentar la prueba de electrones de las estructuras objetivo y se puede utilizar para localizar un objetivo específico de interés.Estas part?...