This work was supported by the Max Planck Society (K.M.D., B.D., A.W.M., T.B., T.B.B., D.J.M., and W.K.), the Cluster of Excellence Frankfurt &#8220;Macromolecular Complexes&#8221; funded by the Deutsche Forschungsgemeinschaft (W.K.) and a postdoctoral EMBO Long-Term Fellowship (V.A.M.G.).

1. Preparation of Mitochondria from Cells or Tissues by Differential Centrifugation
This section describes a general procedure for the isolation of intact mitochondria from various eukaryotic organisms. The precise buffer components and centrifugation speeds need to be optimized for each tissue/species studied.
<ol>
	<li>Break cells in isotonic buffer (e.g.,&#160;250 mM sucrose, 10 mM HEPES pH 7.4), using a glass-bead mill (fungal mycelium)28, enzymatic digestion of the cell wall (Saccharomyces cerevisiae)29, a ball-bearing homogenizer (single-cell eukaryotes/culture cells/nematodes)30, or a blender (animal or plant tissues)31.</li>
	<li>Remove cell debris by filtration through muslin followed by low-speed centrifugation (2,000 x g, 4 &#176;C, 10 min).</li>
	<li>Collect supernatant and pellet mitochondria by high-speed centrifugation (9,000 x g, 4 &#176;C, 10 min).</li>
	<li>Where necessary, use an isotonic density step gradient for additional purification of the mitochondrial fraction32.</li>
</ol>
2. Preparation of Mitochondria for Electron Cryo-tomography
The following section describes how to obtain frozen-hydrated samples for cryo-ET. NOTE: The method involves the use of extremely cold liquid nitrogen and ethane, which can cause severe skin burns. Safety goggles and cryo-protection gloves must be worn. Liquid ethane, which is also flammable, must be handled in a fume hood.
<ol>
	<li>Resuspend pelleted mitochondria in 250 mM trehalose, 10 mM HEPES buffer at pH 7.4 to a concentration of approximately 5 mg/ml total protein.</li>
	<li>Glow-discharge holey carbon EM grids, carbon side up, in a vacuum device according to manufacturer&#8217;s instructions.</li>
	<li>Liquefy a few milliliters of ethane by directing a stream of ethane gas onto the inner side of a liquid nitrogen cooled aluminum container.</li>
	<li>Mix protein A-conjugated gold fiducial suspension 1:1 with mitochondrial suspension and immediately apply 3 &#181;l to a glow-discharge EM grid held in tweezers.</li>
	<li>Place the tweezers in a vitrification device, e.g., a home-made guillotine. Blot off excess liquid with a wedge of filter paper (~5 sec or until liquid stops spreading) and immediately plunge the grid into liquid ethane by releasing the trigger.</li>
	<li>Transfer the grid from liquid ethane into liquid nitrogen. During transfer, remove excess ethane from the grid with filter paper. Place the tip of a wedge-shaped piece of filter paper into the liquid ethane. As the liquid ethane rises, gently drag the grid up the filter paper but keep it below the liquid front. Liquid ethane is removed from the grid by capillary action and once all liquid ethane has been removed, immediately transfer the grid into liquid nitrogen.</li>
	<li>Place the vitrified grid into a grid storage box, and store under liquid nitrogen for later use as appropriate.</li>
</ol>
3. Recording of Tomographic Tilt Series
The following section describes how to set up and collect a tomographic tilt series of a mitochondrion with a Polara electron microscope equipped with a post-column energy filter and CCD camera. Similar protocols are used with all electron cryo-microscopes equipped with CCD or direct electron detector cameras.
<ol>
	<li>Align the Microscope
	<ol>
		<li>Insert test specimen e.g., graphite and gold islands on holey carbon film.</li>
		<li>Select search mode in the microscope&#8217;s low-dose system.</li>
		<li>Bring sample to eucentric height. This is the point of minimal xy movement when tilting the specimen holder. Center a point of interest at 0&#176; tilt, tilt the stage to 20&#176; and re-center the point by altering the z-height. Return to 0&#176; and repeat until lateral offset is minimized.</li>
		<li>Select exposure mode. Choose desired magnification for collecting a tomogram (e.g., 25,000X on detector &#8776; 0.6 nm specimen pixel size).</li>
		<li>Choose a small condenser aperture (50-70 mm) and select a spot size and beam intensity so that the beam is just wider than the imaging device and gives a pixel reading of 60 e-/pixel (CCD) or 14 e-/pixel/sec (direct electron detector, counting mode).</li>
		<li>Center condenser aperture.</li>
		<li>Find Gaussian focus e.g., point of minimal contrast. Reset microscope defocus reading and correct pivot points and rotation center according to manufacturer&#8217;s instructions.</li>
		<li>Dial in desired defocus for recording tomogram. NOTE: High defocus (8 &#181;m) increases contrast but reduces resolution, whereas low defocus (2-4 &#181;m) increases resolution at the expense of contrast.</li>
		<li>Over an empty hole, generate a new gain reference and align energy filter according to manufacturer&#8217;s instructions.</li>
		<li>Align search and exposure modes. In exposure mode, center a point of interest and switch to search mode. Select magnification of 1,500X (0.033 &#181;m/pixel of specimen on detector) and defocus of 100 &#181;m (for increased contrast). Bring point of interest back to center using image shift coils.</li>
		<li>In search mode, adjust spot size and beam intensity so that the beam is just wider than the imaging device and gives a pixel reading of ~20 e-/pixel (CCD) or ~8 e-/pixel/sec (direct electron detector, counting mode).</li>
	</ol>
	</li>
	<li>Finding a Good Specimen Area
	<ol>
		<li>Insert the grid with frozen-hydrated mitochondria into the electron microscope at liquid nitrogen temperature (refer to EM manufacturer&#8217;s instructions).</li>
		<li>In search mode, search the grid for areas of appropriate ice thickness and specimen quality. Take a 6 sec search image of promising areas to determine suitability for tomogram collection. Both the inner and outer mitochondrial membrane should be visible at this magnification.</li>
	</ol>
	</li>
	<li>Recording of a Tomographic Tilt Series
	<ol>
		<li>Once a good specimen area is found, tilt the stage &#177;60&#176; to determine the maximum tilt range that is available without any obstruction of the exposure or focus area by grid bars or ice lumps.</li>
		<li>On a nearby ice-filled hole of similar appearance, change to exposure mode and adjust the beam intensity or image acquisition time so each recorded image has an electron dose of 30-50 e-/pixel for CCDs or 6-8 e-/pixel/s direct electron detectors, counting mode.</li>
		<li>Calculate the dose distribution ratio (I0/I60) by dividing the average electron count for a 1 sec image acquired at 0&#176; with that of a 60&#176; image. This ratio describes the increase in exposure time required to maintain a constant electron count per image with increasing tilt angle (exposure time = 1/cos(&#945;)n where (I0/I60)=2n). The ratio also serves as a good indication of ice thickness. Good tomograms of mitochondria are usually recorded with an I0/I60 = 2.3-2.6.</li>
		<li>Over an empty hole, acquire a 1 sec image in exposure mode and note the electron count per &#197;2. Taking into account the dose distribution ratio, calculate the total number of images that can be recorded for a specific total electron dose (e.g., &#60;40 e-/&#197;2 for structure determination &#38; ~160 e-/&#197;2 for morphology).</li>
		<li>Determine the appropriate tilt interval for tomogram collection by dividing the total tilt range (e.g., 120&#176; for &#177;60&#176;) by the total number of images calculated in 3.3.4.</li>
	</ol>
	</li>
	<li>Set up and record a tomogram with the parameters determined above using appropriate automatic data collection software33,34. Tilt series are usually started at &#177;20&#176; and go through 0&#176; before reaching high tilts in order to maximize the information contents of low-tilt images which is destroyed by increasing electron dose.</li>
</ol>
4. Creation and Segmentation of Tomographic Volumes
This section describes how tomographic volumes of mitochondria are generated from tilt series and how the volumes are present for general viewing.
<ol>
	<li>Save the tomographic tilt series to an appropriate directory. Generate an image stack and convert to an appropriate file format with open-source software, such as dm2mrc or tif2mrc (IMOD package), which convert .dm3, .dm4 or .tif files to mrc stacks. mrc stacks are required for tomographic reconstruction with IMOD35. Other packages require different formats.</li>
	<li>Align the images and generate a tomogram by following the steps detailed in the IMOD tutorial (<a href="http://bio3d.colorado.edu/imod/doc/etomoTutorial.html">http://bio3d.colorado.edu/imod/doc/etomoTutorial.html</a>).</li>
	<li>Enhance the contrast of the tomogram using the non-linear anisotropic diffusion filter distributed with IMOD. This filter works well for membranes and membrane-associated particles such as ATP synthase.</li>
	<li>For visualization, manually segment the tomogram using commercially available programs e.g., Amira. Assign voxels corresponding to the inner or outer membrane and generate a surface. Using the clicker option in the EM-package plugin for AMIRA36 mark the location of ATP synthase particles.</li>
</ol>
5. Subtomogram Averaging of ATP Synthase Dimers and Fitting of X-Ray Structures
The following section describes how subtomogram averages of ATP synthase dimers can be obtained.
<ol>
	<li>Using the marked particles as input and an appropriate software package such as the &#39;Particle Estimation for Electron Tomography&#39; program, calculate a subtomogram average.</li>
	<li>For a resolution estimate, compare two independently determined subtomogram averages by Fourier shell correlation37.</li>
	<li>If available, dock known X-ray structures into the subtomogram average by rigid body fitting, either manually or using automatic sequential docking routines such as those in the program Chimera38.</li>
</ol>

The authors declare that they have no competing financial interests.

The protocol presented here provides an introduction to cryo-ET and subtomogram averaging of mitochondria, but essentially the same procedure can be applied to any other cell compartment or membrane. To obtain the best possible data, critical steps during the procedure are sample preparation, the plunge freezing process and data acquisition strategy. Sample quality, which is critical for success, depends on an optimized freezing protocol to ensure suitable ice thickness, which is of paramount importance for good image contrast. The optimal data acquisition strategy depends on the instrument and sample. Parameters to be optimized include electron dose per image, tilt scheme and defocus. Acquiring a good tomogram of a good sample makes all further processing steps easier and ensures a satisfactory end result.
Cryo-ET combined with subtomogram averaging and atomic model fitting provides details of how protein complexes are arranged in their native cellular environment. The technique is equally suited for investigating the structure of large membrane protein complexes, such as respiratory chain supercomplexes (1.7 MDa), ATP synthase dimers (2x500 kDa), or the nuclear pore complex (~120 MDa)8,9,17. The organization of ATP synthase dimers into rows cannot be observed by high-resolution techniques such as X-ray crystallography, NMR or single-particle cryo-EM, because the dimer rows are disrupted by detergent extraction, which is a necessary step in the isolation and purification of membrane protein complexes.
The arrangement of the ATP synthase in rows of dimers along cristae ridges is a universal organizing principle of mitochondria in all species. The proton-pumping complexes of the electron transport chain, in particular complex I (NADH dehydrogenase), are located in the membrane areas either side of the rows8,9. This organization of the respiratory chain has profound impact on mitochondrial bioenergetics. If the dimer rows cannot form due to the absence of dimer-specific protein subunits, the cells exhibit longer generation times and reduced cellular fitness, as observed in the yeast mutant shown in Figure 341. With the growing interest in mitochondrial diseases, a detailed understanding of the molecular basis governing mitochondrial ultrastructure and function is of paramount importance. Electron cryo-tomography provides a link between protein structure determined by high-resolution methods, and the distribution and arrangement of these proteins in the membrane on the scale of nanometers. This makes cryo-ET an essential tool for understanding mitochondrial structure and function in health and disease.
Further technical developments and improvements in cryo-ET include protein-labeling strategies to identify the position of protein subunits in macromolecular complexes or the location of smaller or less distinct proteins (&#60;0.5 MDa) in cells. In addition, hybrid EM processing methods, which combine subtomogram averaging with single particle analysis or helical reconstruction have recently determined protein structures to ~8 &#197;42,43. These processing methods are currently restricted to purified proteins, which are well separated in ice or form helical assemblies and are currently not applicable to crowded cellular environments like mitochondria and cristae membranes. For data collection, new computing and hardware tools are being developed to enable automated tomogram acquisition, increase image contrast and reduce total required electron dose. The only fundamental limitation in cryo-ET is radiation damage to the sample by the electron beam. This means that only very low electron doses can be used to record each image of a tomographic tilt series, resulting in poor signal-to-noise ratio that ultimately limits the achievable resolution. The new direct electron detectors, released less than a year ago, are currently revolutionizing the field of single-particle cryo-EM44,45. These new detectors provide higher contrast and better resolution at lower electron doses. For electron cryo-tomography, this means that tilt series with smaller step sizes or even dual axis tomograms can be collected without concerns about excessive radiation damage (one CCD image is equivalent in electron dose to 5 direct electron detector images). The vast amounts of data produced by these detectors create their own challenges in data handling, processing and storage, which will have to be overcome.
In addition, phase plates, which work on similar principles as those used routinely in light microscopy to enhance phase contrast, are currently being developed for transmission electron microscopy46,47. This should allow tomograms to be collected closer to focus and therefore at higher resolution, while at the same time preserving low-resolution features necessary for alignment and interpretation of tomographic volumes. Taken together, these technological advances will greatly expand the range of biological questions that can be addressed by cryo-ET.

Mitochondria are the power-houses of the cell. By converting an electrochemical proton gradient across the inner mitochondrial membrane into chemical bond energy, the mitochondrial ATP synthase produces most of the ATP that drives cellular processes. In order to understand the mechanisms behind mitochondrial energy conversion, we need to determine the structure of the ATP synthase in situ, and to find out how it is arranged and distributed in the inner mitochondrial membrane. Although high-resolution structures of most of the mitochondrial ATP synthase components1-3 and low-resolution maps of the whole complex4 are available, it is important to establish the structure and conformation of the working enzyme in the membrane. The distribution of the ATP synthase in the inner mitochondrial membrane has been widely assumed to be random, but an early finding5 and our own initial results6 indicated that this is not the case. Subsequent studies from our group and others7 have confirmed that the ATP synthase is arranged in long rows of dimers along the tightly curved ridges of the inner mitochondrial membrane cristae8, while the proton pumps of the electron transport chain appear to be located at either side of the rows9. This arrangement has important implications for the mechanisms of mitochondrial energy conversion.
The technique we have used to determine this arrangement is electron cryo-tomography (cryo-ET). Cryo-ET is currently the only method that delivers accurate three-dimensional (3D) volumes of cells, cellular compartments or organelles at molecular resolution. Cryo-ET is particularly suitable for studying large complexes in biological membranes, because the membranes appear with good contrast and are easy to trace in 3D tomographic volumes.
Other methods to study the 3D structure of cells or organelles do not provide molecular detail. Super-resolution light microscopy10,11 is superb at revealing the position or distances between light-emitting labels attached to proteins of interest with a precision of tens of nm, but it does not reveal the structure of the protein itself, even at low resolution. Transmission electron microscopy of serial sections12 or block-face imaging by scanning electron microscopy13 of plastic-embedded biological samples provide low-resolution views of cellular volumes but likewise do not reveal molecular detail. Atomic force microscopy14 can in principle deliver molecular or even atomic resolution, but only at the surface of objects on an atomically flat, solid support. Finally, X-ray tomography15 or scattering of intense X-ray pulses from free-electron lasers16 is unlikely to reveal the structure of large, complex, aperiodic objects such as whole cells or organelles at molecular resolution in the foreseeable future. Thus at present, there is no alternative to cryo-ET for studies of the 3D structure of cells or organelles at nanometer resolution.
Cryo-ET is the method of choice for examining the structure and conformation of membrane-associated protein assemblies including the nuclear pore complex17, the influenza spike complexes18, and the flagella motor proteins22,23 but also the organization of whole bacterial cells19 and entry of pathogenic viruses such as HIV in to cells20-23. Cryo-ET is invaluable for visualizing filamentous proteins and their interactions in the cell, including actin filaments24 or axonemes25. The resolution can be enhanced to 2 nm or better by subtomogram averaging26, whereby subvolumes of repeated, regular features are cut out of a tomographic volume and averaged by single-particle image processing techniques.
Cryo-ET involves the acquisition of a series of projection images of a thin specimen (&#60;250nm) taken at different tilt angles in a transmission electron microscope (TEM). The specimen must be thin so that electrons, which interact strongly with matter, are scattered no more than once. Multiple scattering makes the resulting images difficult to interpret and reduces contrast. Images of the selected specimen area are aligned relative to each another and projected into a 3D space by a suitable computer program, generating a 3D volume of the specimen. The alignment of the images is aided by gold fiducial markers, which are mixed with the sample prior to freezing. Ideally 10 or more evenly distributed fiducial markers should be present in each image to achieve a good alignment.
To observe molecular detail, samples are plunge-frozen in liquid ethane, which preserves their native hydrated state. Freezing in liquid ethane is so fast (~105 &#176;C/sec)27 that water does not crystallize but remains in a vitrified, glass-like state. Ice crystal formation damages sensitive biological structures. As biological samples suffer from radiation damage, there is a limit to the total number of scattering events the specimen can tolerate. Images are thus acquired in low-dose mode: An area of interest is identified at low magnification (1,500X) with an electron dose below 1 e-/nm2 (search mode). The image is then focused at a higher magnification, off the area of interest (focus mode). Only when an image is acquired, the area of interest is irradiated with a higher electron dose (exposure mode).
Here, we present an overview of how to collect and process electron cryo-tomograms, using ATP synthase dimers in the inner mitochondrial membrane as an example. The following protocol describes how to prepare mitochondria for cryo-ET, how to set up and collect a tilt series with a specific total electron dose, and how to process the tilt series to obtain a 3D volume of the area of interest. An overview of the procedure is illustrated in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" width="961">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sucrose</td>
			<td style="width:235px;">ROTH</td>
			<td style="width:119px;">4621.2</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trehalose</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">HEPES</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Zymolase</td>
			<td style="width:235px;">biomol</td>
			<td style="width:119px;">Z1000.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Density gradient medium</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1556 or P1644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Protein A gold</td>
			<td style="width:235px;">Aurion</td>
			<td style="width:119px;">810.111</td>
			<td>10 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Ethane</td>
			<td style="width:235px;">Air Liquide</td>
			<td style="width:119px;">P0502S02R0A001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Nitrogen</td>
			<td style="width:235px;">Air Liquide</td>
			<td style="width:119px;">I4150RG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Consumables</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Filter paper</td>
			<td style="width:235px;">Whatman, GE Healthcare</td>
			<td style="width:119px;">1004 090</td>
			<td>Nr 4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tweezers</td>
			<td style="width:235px;">Dumont</td>
			<td style="width:119px;">T506</td>
			<td>Non-magnetic, pattern #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EM combined test grid</td>
			<td style="width:235px;">Plano GmbH</td>
			<td style="width:119px;">S142</td>
			<td>grapite and gold islands on holey carbon film</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Holey carbon grids</td>
			<td style="width:235px;">Quantifoil Micro Tools GmbH or C-flat</td>
			<td style="width:119px;"></td>
			<td>R2/2 300 Cu mesh&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cryo grid boxes</td>
			<td style="width:235px;">Plano GmbH</td>
			<td style="width:119px;">160-42</td>
			<td>Alternative home-made metal boxes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Equipment</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Potter</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7984</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blender</td>
			<td style="width:235px;">Waring</td>
			<td style="width:119px;">8011S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glass bead mill - bead beater</td>
			<td style="width:235px;">BioSpec Products</td>
			<td style="width:119px;">1107900-105</td>
			<td>Including 0.5 mm beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Ball-bearing (Balch) homogeniser</td>
			<td style="width:235px;">isobiotec</td>
			<td style="width:119px;">H8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:235px;">Thermo Scientific</td>
			<td style="width:119px;">46915</td>
			<td>Sorvall RC6+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glow-discharge apparatus</td>
			<td style="width:235px;">Bal-Tec A.G.</td>
			<td style="width:119px;">CTA 010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Grid vitrification device</td>
			<td style="width:235px;">FEI, Gatan or Leica</td>
			<td style="width:119px;"></td>
			<td>Alternative home-made plunger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Transmission electron microscope</td>
			<td style="width:235px;">FEI or Jeol</td>
			<td style="width:119px;"></td>
			<td>300 keV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Direct electron detector (CMOS)</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>4k x 4k pixels, counted mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Charge coupled device</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>4K x 4K pixels (alternative to Direct electron detector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Energy filter</td>
			<td style="width:235px;">Gatan or Jeol</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Software</td>
			<td style="width:235px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Image acquisition software</td>
			<td style="width:235px;">Gatan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tomogram acquisition software</td>
			<td style="width:235px;">Gatan, FEI, USCF, or UC-Boulder</td>
			<td style="width:119px;"></td>
			<td>4 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tomogram processing software</td>
			<td style="width:235px;">FEI, UCSF or UC-Boulder</td>
			<td style="width:119px;"></td>
			<td>3 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Subtomogram averaging software</td>
			<td style="width:235px;">UC-Boulder, or University of Basel</td>
			<td style="width:119px;"></td>
			<td>2 alternatives</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Tomogram visualisation software</td>
			<td style="width:235px;">UC-Boulder, FEI, or University of Utah</td>
			<td style="width:119px;"></td>
			<td>3 alternatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">visualisation software plugin</td>
			<td style="width:235px;">Goethe university</td>
			<td style="width:119px;"></td>
			<td>http://www.biophys.uni-frankfurt.de/frangakis/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Structure visualisation software</td>
			<td style="width:235px;">UCSF</td>
			<td style="width:119px;"></td>
			<td>http://www.cgl.ucsf.edu/chimera/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Numerical calculation software</td>
			<td style="width:235px;">MathWorks</td>
			<td style="width:119px;"></td>
			<td>http://www.mathworks.de/academia/</td>
		</tr>
	</tbody>
</table>

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.

Electron cryo-tomograms of mitochondria clearly reveal the 3D morphology of the organelle (Figure 2). Manual segmentation of the membranes in a tomographic volume illustrates the structure of the cristae in a mitochondrion. By imaging mitochondria from different yeast knockout strains that lack certain protein components, the effect of these proteins on cristae morphology can by assessed. Figure 3 shows a mitochondrion from a yeast strain lacking ATP synthase subunit e. This component of the ATP synthase complex is required for the dimerization of the mitochondrial ATP synthase. Mitochondria from this strain lack the normal lamellar cristae of the wildtype mitochondria (Figure 2) and instead contain a number of inner membrane compartments. These compartments are either devoid of cristae or contain small balloon-shaped membrane invaginations (Figure 3).
In tomograms with good contrast, large mitochondrial protein complexes, in this case ATP synthase dimers, are easily visible (Figure 4; Movie 1). The structures of the complexes can be determined at 2-3 nm resolution by subtomogram averaging (Figure 5; Movie 2). The average volumes may be placed back into the tomogram in order to assess the organization of individual complexes relative to each other and to other protein complexes in the membrane (Figure 6; Movie 3).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig1highres.jpg" width="500" /> 
Figure 1. Flow-chart showing the stages of electron cryo-tomography . <a href="https://www.jove.com/files/ftp_upload/51228/51228fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig2highres.jpg" width="500" /> 
Figure 2. Morphology of a mitochondrion from wildtype S. cerevisiae. Central slice through a tomographic volume of a wildtype S. cerevisiae mitochondrion (left) and corresponding surface-rendered volume (right). The segmented volume of the outer membrane is shown in grey and the volumes of the inner boundary and cristae membranes in light blue. Adapted from Davies et al8. <a href="https://www.jove.com/files/ftp_upload/51228/51228fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig3highres.jpg" width="500" /> 
Figure 3. Mitochondrion from a S. cerevisiae strain lacking a subunit required for ATP synthase dimerization. Slice through tomographic volume (left) and accompanying surface-rendered volume (right) of a mitochondrion from a S. cerevisiae strain lacking the protein subunit e required for ATP synthase dimerization. When compared with Figure 2, the mitochondrion from the mutant strain lacks the normal lamellar cristae of wildtype mitochondria. Instead, the mitochondrion has many inner membrane compartments with either no cristae or balloon-shaped cristae. Thus electron cryo-tomography highlights alterations in membrane morphology due to gene deletions. Adapted from Davies et al8. <a href="https://www.jove.com/files/ftp_upload/51228/51228fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig4highres.jpg" width="500" /> 
Figure 4. Mitochondrion from the fungus P. anserina. Slice through tomographic volume (left) and accompanying surface-rendered volume (right) of a mitochondrion from the filamentous fungus P. anserina. In this tomogram, rows of 10 nm particles (yellow arrowheads) are located above highly curved membrane ridges in the inner membrane cristae (see Movie 1). These particles were identified as ATP synthase dimers by subtomogram averaging. From Davies et al9. <a href="https://www.jove.com/files/ftp_upload/51228/51228fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig5highres.jpg" width="500" /> 
Figure 5. Structure and organization of the mitochondrial ATP synthase. Side and top view showing the electron density of an ATP synthase dimer from S. cerevisiae as determined by subtomogram averaging with fitted atomic models (left). Mitochondrial inner membrane vesicle showing the organization of ATP synthase dimers in rows (right). The figure was generated by positioning the subtomogram average of the ATP synthase dimer into the segmented volume of the membrane vesicle, using the coordinates calculated during averaging.&#160; Adapted from Davies et al8. Atomic models: F1/rotor-ring [PDB:2WPD]39 (blue and purple); oligomycin sensitive conferring protein-OSCP [PDB:2BO5]40 (green); peripheral stalk fragment [PDB:2CLY]1 with N-terminal residues from [PDB:2WSS]2 (yellow and red) (see Movie 2). <a href="https://www.jove.com/files/ftp_upload/51228/51228fig5highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51228/51228fig6highres.jpg" width="500" /> 
Figure 6. Isolated crista vesicle from a P. anserina mitochondrion. Slices from a tomographic volume (left) and accompanying surface-rendered volume (right) of a crista vesicle from P. anserina. Protein densities protruding from the membrane are clearly visible. The densities indicated by yellow arrowheads are ATP synthase dimers, as identified by subtomogram averaging. Green arrowheads point to densities identified by antibody labeling as NADH dehydrogenase (complex 1; for details see9). Segmentation of the protein densities reveals their organization in the cristae, with the ATP synthase dimers (red and yellow) forming rows along the highly curved cristae ridges and the NADH dehydrogenase complexes (green) in the membrane regions on either side of the rows (see Movie 3).&#160; From Davies et al9. <a href="https://www.jove.com/files/ftp_upload/51228/51228fig6highres.jpg" target="_blank">Click here to view larger image.</a>
Movie 1. Electron cryo-tomogram of a P. anserina mitochondrion. The movie shows successive slices through a tomographic volume taken of a mitochondrion from the filamentous fungus P. anserina. Rows of ATP synthase are indicated by yellow arrowheads. The surface-rendered, segmented volume shows the location of ATP synthase (yellow spheres) in relation to the 3D cristae structure. Outer membrane, grey; inner boundary membrane, transparent blue; cristae membranes, opaque blue. From Davies et al9. See also Figure 4. <a href="https://www.jove.com/files/ftp_upload/51228/movie1.mp4" target="_blank">Click here to view video.</a>
Movie 2. Subtomogram average of the mitochondrial ATP synthase dimer from S. cerevisiae with fitted atomic models. The average was calculated from 121 subvolumes. The density is displayed at three contour levels: 1s - mesh, 2s - light grey and 3s - dark grey. Atomic models were fitted into the density using the sequential fit routine in Chimera. Atomic models: F1/rotor-ring [PDB:2WPD]39 (blue and purple); oligomycin sensitive conferring protein-OSCP [PDB:2BO5]40 (green); peripheral stalk fragment [PDB:2CLY]1 with N-terminal residues from [PDB:2WSS]2 (yellow and red). <a href="https://www.jove.com/files/ftp_upload/51228/movie2.mov" target="_blank">Click here to view video.</a>
Adapted from Davies et al8. See also Figure 5.
Movie 3. Isolated cristae vesicle from a P. anserina mitochondrion. Successive slices through the tomographic volume are shown, followed by the segmented surface-rendered volume. Protein densities protruding from the membrane are clearly visible. Red and yellow densities are ATP synthase dimers. Green densities are NADH dehydrogenase (complex I) as determined by antibody labeling. From Davies et al9. See also Figure 6. <a href="https://www.jove.com/files/ftp_upload/51228/movie3.mpg" target="_blank">Click here to view video.</a>

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

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 ...

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29.4K Views.  Max Planck Institute of Biophysics. 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.

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

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

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 ...

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

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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