The studies were performed in the EM core at the Institute of Cellular and Organismic Biology and the cryo-EM core of Academia Sinica, Taipei, Taiwan. The work was supported by Academia Sinica and MOST.

1. Section Drosophila Tissues Using a Vibrating Blade Microtome
<ol>
	<li>Anesthetize Drosophila on ice and immerse each single fly in 1 mL of 4% low melting agarose in phosphate buffer. Allow agarose to solidify on ice. Typically, 4 - 6&#160;flies were processed.</li>
	<li>Use a vibrating blade microtome to section agarose gel embedded Drosophila into slices with 100 &#181;m thickness and immerse in fixative solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer. 
	NOTE: Vibratome sectioning is preferred because tissue architecture remains more intact compared to other methods. Alternatively, dissecting tweezers can be used to dissect IFM into the fixative solution containing 2.5% glutaraldehyde in 0.1 M phosphate buffer.</li>
</ol>
2. Prepare EM Specimens by the High-pressure Freezing and Freeze Substitution (HPF/FS) Method
<ol>
	<li>Wash tissue sections in 3 drops (~150 &#181;L) of phosphate buffer, followed by 2 drops (~100 &#181;L) of phosphate buffer with 20% BSA. Then place sections in gold carriers for HPF filled with buffer and 20% BSA.</li>
	<li>Load the sample containing carriers into a high-pressure freezer according to the user manual.</li>
	<li>After freezing, release carriers from the holder under liquid nitrogen and transfer to a freeze-substitution device cooled to -140 &#176;C.</li>
	<li>Perform the freeze-substitution protocol as shown in Table 1, with the FS cocktail containing 2% glutaraldehyde, 2% osmium tetroxide, and 0.1% uranyl acetate in acetone.</li>
	<li>Carefully remove the specimens from the carriers with a needle and embed specimens in resin at room temperature. Polymerize the resin at 65 &#176;C for 16 h. 
	NOTE: FS protocols should be modified to prepare other types of samples. HPF/FS is preferred to preserve the ultrastructure and minimize the loss of cellular contents.
	<ol>
		<li>Alternatively, apply a chemical fixation protocol. Fix specimens with 2.5% glutaraldehyde overnight, wash with buffers and then fix with 1% osmium tetroxide for 2 h. Wash and dehydrate with ascending concentrations of ethanol and then infiltrate and embed specimens in Spurr&#39;s resin before polymerizing at 65 &#176;C for 16 h.</li>
	</ol>
	</li>
</ol>
3. Prepare Serial-sections of the Specimens for Electron Tomography
<ol>
	<li>Trim the specimen blocks to expose the desired block face that contains tissue.</li>
	<li>Pretreat gold particles (10 nm in diameter) with 1% BSA for 30 min. Wash and suspend gold particles in PBS buffer. Overlay gold particles on copper slot grids coated with carbon film to create fiducial markers.</li>
	<li>Check under TEM to have sufficient fiducial markers (at least 5 - 10 markers) in the field of view of tomography acquisition.</li>
	<li>Cut serial sections, 200 - 250 nm in thickness, using an ultramicrotome.</li>
	<li>Collect serial sections on slot grids using a perfect loop for thin sections.</li>
	<li>Stain the sections with Reynold&#39;s lead citrate for 10 min.</li>
	<li>Overlay a second layer of fiducial gold particles on the top of the sections.</li>
</ol>
4. Collect Double-tilt Electron Tomography
<ol>
	<li>Load the grid onto a dual-axis tomography holder and insert into the transmission electron microscope operating at 200 kV.</li>
	<li>Align the microscope at the eucentric focus.</li>
	<li>Set up the automatic data collection software. Adjust and align the electron beam at the multi-scale imaging setting. Refer to user manual for operation detail10 (Figure 2).</li>
	<li>Acquire camera dark and bright references in an empty area without carbon film under the tomography collection setting.</li>
	<li>Collect a grid atlas at low magnification. Select mitochondria on serial sections as targets for tomography collection.</li>
	<li>Acquire a tilt series from -60&#176; to +60&#176; with 2&#176; increments on axis-A for each target. 
	NOTE: The tilt angles will be mechanically constrained by the design of the specimen holder. The holder will block the beam at high tilt angles.</li>
	<li>To collect the second tilt series, rotate the specimen holder 90&#176;. Acquire a new atlas. Select corresponding positions and acquire the tilt series on axis-B for each target. 
	NOTE: Other software packages, such as SerialEM and Xplore3D, are available for automatic data collection.</li>
</ol>
5. Reconstruct 3D Tomograms and Segment Sub-volumes Using Software
<ol>
	<li>Reconstruct tomograms of dual-axis tilt images using IMOD software11.
	<ol>
		<li>Refer to user manual for operation detail.</li>
		<li>Align the individual tilt series by the positions of gold fiducial markers. Reconstruct tomograms either by the Back Projection method or by the Simultaneous Iterative Reconstruction Technique (SIRT) method for both axis-A and axis-B, respectively (Figure 3).</li>
		<li>Combine tomograms of axis-A and axis-B to generate a double-tilt tomogram with reduced missing wedge artifact.</li>
		<li>Join together double-tilt tomograms of serial sections to obtain reconstructions covering entire mitochondrion volume. Model the gaps between serial sections by the IMOD program.</li>
		<li>Trim the joined tomograms and bin to a desired size for volume segmentation.</li>
	</ol>
	</li>
	<li>Analyze joined serial tomograms using 3D software.
	<ol>
		<li>Refer to user manual for operation detail.</li>
		<li>Filter the tomograms with a Gaussian filter (or other desired method) to improve the contrast of features and reduce background densities.</li>
		<li>Segment the ultrastructure of mitochondria either manually or automatically.</li>
		<li>Display segmentation models to allow inspection in 3D.</li>
		<li>Generate movies using tools available in 3D.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Dahl, R., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+the+preservation+of+biological+structure:+theory+and+practice.">High-pressure freezing for the preservation of biological structure: theory and practice.</a> J Electron Microsc Tech. 13 (3), 165-174 (1989).</li><li>Sabatini, D. D., Bensch, K., Barrnett, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemistry+and+electron+microscopy.+The+preservation+of+cellular+ultrastructure+and+enzymatic+activity+by+aldehyde+fixation.">Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.</a> J Cell Biol. 17, 19-58 (1963).</li><li>Rigort, 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=Micromachining+tools+and+correlative+approaches+for+cellular+cryo-electron+tomography.">Micromachining tools and correlative approaches for cellular cryo-electron tomography.</a> J Struct Biol. 172 (2), 169-179 (2010).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biol. 2 (11), e329 (2004).</li><li>Lucic, V., Forster, F., 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=Structural+studies+by+electron+tomography:+from+cells+to+molecules.">Structural studies by electron tomography: from cells to molecules.</a> Annu Rev Biochem. 74, 833-865 (2005).</li><li>Soto, G. E., 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=Serial+section+electron+tomography:+a+method+for+three-dimensional+reconstruction+of+large+structures.">Serial section electron tomography: a method for three-dimensional reconstruction of large structures.</a> Neuroimage. 1 (3), 230-243 (1994).</li><li>Jiang, Y. F., 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=Electron+tomographic+analysis+reveals+ultrastructural+features+of+mitochondrial+cristae+architecture+which+reflect+energetic+state+and+aging.">Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging.</a> SciRep. 7, 45474 (2017).</li><li>Cogliati, S., Enriquez, J. A., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Cristae:+Where+Beauty+Meets+Functionality.">Mitochondrial Cristae: Where Beauty Meets Functionality.</a> TrendsBiochemSci. 41 (3), 261-273 (2016).</li><li>Friedman, J. R., Nunnari, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+form+and+function.">Mitochondrial form and function.</a> Nature. 505 (7483), 335-343 (2014).</li><li>Suloway, C., 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=Fully+automated,+sequential+tilt-series+acquisition+with+Leginon.">Fully automated, sequential tilt-series acquisition with Leginon.</a> J Struct Biol. 167 (1), 11-18 (2009).</li><li>Mastronarde, D. N., Held, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tilt+series+alignment+and+tomographic+reconstruction+in+IMOD.">Automated tilt series alignment and tomographic reconstruction in IMOD.</a> J Struct Biol. 197 (2), 102-113 (2017).</li></ol>

The authors declare that they have no competing financial interests.

In this protocol, we describe an optimized workflow for applying serial-section electron tomography to study 3D mitochondrial ultrastructure of Drosophila indirect flight muscle. Preservation of ultrastructure in the sample is the primary technical challenge for this type of analysis. In order to best preserve ultrastructure two methodological steps were included. First, tissue was sampled by sectioning with vibrating blade microtome in order to maintain the tissue architecture as much as possible. Second, a HPF/FS protocol was optimized to preserve organelle ultrastructure during the preparation of embedded specimen blocks. Specimens were frozen under high pressure, which lowers the freezing point of water and reduces the formation of ice crystals that damage ultrastructure1. Specimens as thick as 0.1 mm can be vitrified instantaneously, and then subjected to freeze substitution to generate specimen blocks for EM analysis. The improved preservation of ultrastructure by HPF/FS was noted, when compared to chemical fixation methods. Using this series of steps for sample preparation, preservation of mitochondrial double membranes and cristae membranes was dramatically improved.
Obtaining serial sections of the specimen is the most challenging step of the method. As the electron beam has limited penetration power, the thickness of the section is restricted to either 250 nm or 500 nm using a TEM operating at 200 kV or 300 kV, respectively. Because the thickness of a mitochondrion may be more than 2 &#181;m, serial sections are required to obtain full volume reconstructions. However, recovering a sufficient number of serial sections to span an entire organelle is a technical challenge. Trimming the block face to be as flat as possible between the bases of the trapezoid can allow a slot grid to accommodate more sections and thus cover larger volumes. Additionally, using a perfect loop for thin sections increases the success rate of transferring the serial sections to the grid.
Serial-section electron tomography can be accomplished with standard EM core equipment. However, the method has some inevitable limitations that arise from technical constraints. One is that material is inevitably lost between serial sections, leaving gaps in the joined reconstruction. Second is the missing wedge artifact, which arises due to the limited tilt angles that are achievable. This restriction occurs because the specimen holder cannot be turned in a full rotation without blocking the electron beam. Despite these limitations, serial-section electron tomography provides sufficient resolution to reveal cellular and organelle ultrastructure in 3D.
For smaller scale imaging, cryo-electron tomography is an emerging technology that can be used to obtain the structure of macromolecular complexes and assemblies in situ at nm or sub-angstrom resolution in combination with sub-tomographic reconstruction3. In this application, cells are thinned by sectioning or by focused ion beam milling under liquid nitrogen. The tomograms are collected under cryo-conditions where molecular structures are preserved close to the native state without chemical fixation, dehydration, or embedding. At the other end of scale, to analyze large tissue volumes at the expense of resolution, serial block-face scanning electron microscopy is an appealing modality even though it requires a specific instrument4.

Electron microscopy is a valuable tool to study the structural context of subcellular assemblies and organelles that perform cellular processes. Methods have been developed to preserve the ultrastructure of tissues or cells either by chemical fixation with aldehydes or by high pressure freezing (HPF) followed by freeze substitution (FS)1,2. The embedded specimen blocks may then be sectioned, stained, and observed with a transmission electron microscope (TEM). The HPF specimen could also be processed under cryo-condition, such as by cryo-sectioning or by focused ion beam (FIB) milling, and observed by cryo-EM3,4.
Although thin-section EM provides informative morphological insights, the resulting 2D images can only reveal the ultrastructure of a particular cross-section. How ultrastructure is organized in a 3D volume remains obscured. In order to visualize cellular ultrastructure in three dimensions, an electron tomography method was developed where series of tilt images were acquired and back projected to generate a tomographic reconstruction5 (Figure 1). A double tilt series can be collected by rotating the sample 90&#176; and acquiring a second tilt series. This will minimize missing-wedge artifacts that result from limited sampling angles and improve resolution of the tomogram.
Here, we describe the application of serial-section electron tomography to study mitochondrial ultrastructure of Drosophila indirect flight muscle (IFM)6,7,8,9. In order to obtain 3D reconstructions covering entire mitochondria (approximately 2.5 &#181;m-thick), serial sections were obtained from Drosophila IFM tissue blocks. Tomograms of each section were collected individually using automatic data collection software. Tomographic reconstructions were generated and serial tomograms were joined with the IMOD package to obtain a reconstructed volume of an entire mitochondrion. The joined tomograms were analyzed by 3D software. The densities of mitochondrial cristae were segmented to generate a segmentation model that revealed the organization in three-dimensions.

<table width="852">
	<tbody>
		<tr height="40">
			<td height="40" style="width:252px;height:40px;">vibrating blade microtome</td>
			<td style="width:123px;">Leica&#160;</td>
			<td style="width:267px;">VT1200S</td>
			<td style="width:212px;">Tissue sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">high-pressure freezer</td>
			<td>Leica&#160;</td>
			<td>EM HPM100</td>
			<td>Specimen preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">freeze-substitution device</td>
			<td>Leica&#160;</td>
			<td>EM AFS2</td>
			<td>Specimen preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ultramicrotome</td>
			<td>Leica&#160;</td>
			<td>EM UC7</td>
			<td>Ultra-thin sectioning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">dual-axis tomography holder</td>
			<td>Fischione&#160;</td>
			<td>Model 2040</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">transmission electron microscope</td>
			<td>FEI</td>
			<td>&#160;Tecnai F20</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CCD</td>
			<td>Gatan&#160;</td>
			<td>UltraScan 1000</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leginon</td>
			<td colspan="2" style="width:388px;">NRAMM/AMI</td>
			<td>tomography collection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IMOD</td>
			<td colspan="2" style="width:388px;">Boulder Laboratory for 3-D Electron Microscopy of Cells</td>
			<td>Tomography reconstruction&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Avizo 3D</td>
			<td>FEI</td>
			<td></td>
			<td>Tomography analysis</td>
		</tr>
	</tbody>
</table>

3d mitochondrial ultrastructure of drosophila indirect flight muscle revealed by serial-section electron tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

We applied serial-section electron tomography to analyze structural features of mitochondrial cristae that reflects its energetic state and aging. We showed that mitochondria of Drosophila IFM form an integrated cristae and matrix network in 3D (Figure 4)7. In addition, mutant flies with mitochondrial DNA replication defect and an accelerated aging phenotype accumulated mitochondria that contained subsections of onion-like swirling core (Figure 5)7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56567/56567fig1.jpg" /> 
Figure 1: Illustration of electron tomography reconstruction from a tilt series. In the experiment, an electron beam is passed through a 3D object as the object is tilted to various degrees. For each tilt condition, a 2D projection is generated and captured with a camera. The 2D projections are then back projected to reconstruct the 3D object based on the central slice theorem. In the illustration, the same process is shown for an original 2D image that is projected into a single dimension under various tilt angles. The 1D projections are then used to reconstruct the 2D image. <a href="https://cloudflare.jove.com/files/ftp_upload/56567/56567fig1large.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/56567/56567fig2.jpg" /> 
Figure 2: Automatic data collection. An image viewer display of software showing the atlas of a slot grid containing serial sections, multi-scale targeting of mitochondria and acquired tilt images. Scale bar = 500 &#181;m (left top panel), 100 &#181;m (left bottom panel), 1 &#181;m (right panel). <a href="https://cloudflare.jove.com/files/ftp_upload/56567/56567fig2large.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/56567/56567fig3.jpg" /> 
Figure 3: Serial-section electron tomography of a single mitochondrion in Drosophila indirect flight muscle. (A) 2D micrographs and (B) 3D tomograms from serial sections covering the entire volume of a mitochondrion. (C) The joined serial-section tomograms are projected to create a longitudinal section, with the z-axis shown vertically. Notably, tissue sectioning led to loss of material, leaving gaps between joined tomograms. Scale bar = 500 nm. <a href="https://cloudflare.jove.com/files/ftp_upload/56567/56567fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56567/56567fig4.jpg" /> 
Figure 4: Integrated intra-mitochondria cristae and matrix network revealed in 3D. (A) Slices of mitochondrial electron tomographic reconstructions showing switches between lamellar membranes through the z-axis. (B) Illustrations of the observed cristae switching patterns of right handed and/or left-handed spirals. (C) Tomographic segmentation illustrating a left-handed spiral in 3D (D) Cristae switching patterns were analyzed and color-rendered on the segmentation model (E, F). Tomographic slice showing lateral matrix confluency (dark densities, marked red) across cristae membranes (white densities). (G) Segmentation model of the tomogram in (E) showing lateral matrix confluency (in red) and representative cristae (in gray). Scale bar = 50 nm (A), 200 nm (C), and 300 nm (D, E, F, G). The figure was the reprint from Jiang et al.7 <a href="https://cloudflare.jove.com/files/ftp_upload/56567/56567fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56567/56567fig5.jpg" /> 
Figure 5: Mitochondria with onion-like swirling cores accumulated in flies with mitochondrial DNA replication defects during aging. Representative tomographic slices and the corresponding segmentation (right panels) showing a cross-sectional view (top) and a longitudinal-section view (bottom) of a swirling core. Volume segmentation is indicated by arbitrary color rendering to highlight the swirling core. Scale bar: 200 nm. The figure was the reprint from Jiang et al.7 <a href="https://cloudflare.jove.com/files/ftp_upload/56567/56567fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td colspan="2">Temp</td>
			<td colspan="2">Time</td>
			<td colspan="2">Solution</td>
		</tr>
		<tr>
			<td>1</td>
			<td colspan="2">-140 &#176;C to -9 0&#176;C</td>
			<td colspan="2">30 min</td>
			<td colspan="2">Liquid nitrogen</td>
		</tr>
		<tr>
			<td>2</td>
			<td colspan="2">-90 &#176;C</td>
			<td colspan="2">96 hr</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>3</td>
			<td colspan="2">-90 &#176;C to -60 &#176;C</td>
			<td colspan="2">6 hr (5 &#176;C/hr)</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>4</td>
			<td colspan="2">-60 &#176;C</td>
			<td colspan="2">12 hr</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">-60 &#176;C to -25 &#176;C</td>
			<td colspan="2">7 hr (5 &#176;C/hr)</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>6</td>
			<td colspan="2">-25 &#176;C</td>
			<td colspan="2">12 hr</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>7</td>
			<td colspan="2">-25 &#176;C to 0 &#176;C</td>
			<td colspan="2">5 hr (5 &#176;C/hr)</td>
			<td colspan="2">FS cocktail</td>
		</tr>
		<tr>
			<td>8</td>
			<td colspan="2">0 &#176;C</td>
			<td colspan="2">1 hr x 3 times</td>
			<td colspan="2">Acetone</td>
		</tr>
		<tr>
			<td>9</td>
			<td colspan="2">Room temp</td>
			<td colspan="2"></td>
			<td colspan="2">Resin infiltration</td>
		</tr>
	</tbody>
</table>
Table 1: Freeze-substitution protocol.

Watch this Scientific Journal Video about 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography at JoVE.com

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

3d-mitochondrial-ultrastructure-drosophila-indirect-flight-muscle

Nanyang Technological University

Research

JoVE Journal

Biology

8.4K Views.  National Taiwan University. In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

Video: 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

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

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

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

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

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.

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

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

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

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

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

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

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...