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><tbody><tr><td>vibrating blade microtome</td><td>Leica</td><td>VT1200S</td><td>Tissue sectioning</td></tr><tr><td>high-pressure freezer</td><td>Leica</td><td>EM HPM100</td><td>Specimen preparation</td></tr><tr><td>freeze-substitution device</td><td>Leica</td><td>EM AFS2</td><td>Specimen preparation</td></tr><tr><td>ultramicrotome</td><td>Leica</td><td>EM UC7</td><td>Ultra-thin sectioning</td></tr><tr><td>dual-axis tomography holder</td><td>Fischione</td><td>Model 2040</td><td>tomography collection</td></tr><tr><td>transmission electron microscope</td><td>FEI</td><td>Tecnai F20</td><td>tomography collection</td></tr><tr><td>CCD</td><td>Gatan</td><td>UltraScan 1000</td><td>tomography collection</td></tr><tr><td>Leginon</td><td>NRAMM/AMI</td><td>tomography collection</td><td></td></tr><tr><td>IMOD</td><td>Boulder Laboratory for 3-D Electron Microscopy of Cells</td><td>Tomography reconstruction&amp;nbsp;</td><td></td></tr><tr><td>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

Research

JoVE Journal

Biology

8.5K Views.  National Taiwan University. In dit protocol tonen we de toepassing van seriële-sectie Elektronentomografie ophelderen van mitochondriale structuur in Drosophila indirecte vlucht spier.

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

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genetics

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Developmental Biology

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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 ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Neuroscience

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

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 ...

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

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

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

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

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

Samenvatting

Meer video's verkennen