Name: studying the supramolecular organization of photosynthetic membranes within freeze-fractured leaf tissues by cryo-scanning electron microscopy
Uploaded: 2016-06-23T14:00:00
Description: Watch this Scientific Journal Video about Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy at JoVE.com

We thank Andres Kaech (University of Zurich) for his helpful advice on scanning electron microscopy imaging. This work was supported by the United States-Israel Binational Agricultural Research and Development Fund (grant no. US-4334-10, Z.R.), the Israel Science Foundation (grant no. 1034/12, Z.R.), and the Human Frontier Science Program (RGP0005/2013, Z.R.). The electron microscopy studies were conducted at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science.

1. Cryo-fixation of Leaf Tissues by High-pressure Freezing
Note: This section describes how to carry out high-pressure freezing of leaf tissues for a freeze-fracture experiment. For considerations related to plant samples see29. This can be adapted for other types of tissues or samples with some modification.
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	<li>Using the corner of a razor blade, scratch the bottom of the 0.1 mm cavity of a 0.1/0.2 mm aluminum platelet (Figure 1). Prepare at least a dozen p...

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	<li>Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+consequences+of+grana+stacking+of+thylakoid+membranes+in+vascular+plants:+a+personal+perspective.">Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: a personal perspective.</a> Aust. J. Plant Physiol. 26 (7), 625-639 (1999).</li><li>Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+Faces+of+Frozen+Membranes.">Fracture Faces of Frozen Membranes.</a> Proc. Natl. Acad. Sci. U. S. A. 55 (5), 1048-1056 (1966).</li><li>Branton, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-Etching+Nomenclature.">Freeze-Etching Nomenclature.</a> Science. 190 (4209), 54-56 (1975).</li><li>Goodenough, U. W., 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=Structural+Differentiation+of+Stacked+and+Unstacked+Chloroplast+Membranes+-+Freeze-Etch+Electron+Microscopy+of+Wild-Type+and+Mutant+Strains+of+Chlamydomonas.">Structural Differentiation of Stacked and Unstacked Chloroplast Membranes - Freeze-Etch Electron Microscopy of Wild-Type and Mutant Strains of Chlamydomonas.</a> J. Cell Biol. 48 (3), 594-619 (1971).</li><li>Simpson, D. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+Identification+of+the+Exoplasmic+Face+Particles+on+the+Freeze-Fractured+Thylakoid+Membranes+-+a+Study+Using+Double+and+Triple+Mutants+from+Chlamydomonas-Reinhardtii+Lacking+Various+Photosystem-Ii+Subunits+and+the+Cytochrome+B6/F+Complex.">Further Identification of the Exoplasmic Face Particles on the Freeze-Fractured Thylakoid Membranes - a Study Using Double and Triple Mutants from Chlamydomonas-Reinhardtii Lacking Various Photosystem-Ii Subunits and the Cytochrome B6/F Complex.</a> Eur. J. Cell Biol. 59 (1), 176-186 (1992).</li><li>Olive, J., Vallon, O., Wollman, F. 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The technique described in this paper allows investigation of freeze-fractured membranes within the context of well-preserved high-pressure frozen plant tissues by cryo-scanning electron microscopy. The major advantage of using these procedures is that sample preparation is purely physical; no steps involving chemicals or dehydration are necessary. Thus, it allows studying biological structures at a near-native state26,32. The benefit of using leaf tissues is that one can obtain information on the overall cell...

Oxygenic photosynthesis, originating in ancient cyanobacteria, was inherited by algae and land plants by endosymbiotic events that led to development of the chloroplast organelle. In all modern-day oxygenic phototrophs, photosynthetic electron transport and the generation of proton-motive force and reducing power are carried out within flattened sac-like vesicles termed &#39;thylakoid&#39; membranes. These membranes house the protein complexes that carry out the light-driven reactions of photosynthesis and provide a medium for energy transduction. The thylakoid membranes of plants and (some) algae are differentiated into two distinct morphological domains: tightly appressed membrane regions called &#39;grana&#39; and unstacked membranes that interconnect the grana, called &#39;stroma lamellae&#39;1. Various freeze-fracture studies of plant and algal thylakoid membranes have been conducted, starting in the early 1970s. When freeze-fractured, membranes split along their hydrophobic core2, generating an exoplasmic face (EF) and a protoplasmic face (PF), depending on the cellular compartment which the half-membrane borders, as originally coined by Branton et al. in 19753. Plant and algal thylakoids have four different fracture faces: EFs, EFu, PFs and PFu, with &#39;s&#39; and &#39;u&#39; denoting &#39;stacked&#39; and &#39;unstacked&#39; membrane regions, respectively. The membrane protein complexes, which are not split or broken, have the tendency to remain with either the E or P side of the membrane. The initial observations that the different fracture faces of the thylakoids contain particles of different sizes and densities4, and the numerous investigations that followed, led to identification and correlation between the observed particles and the membrane protein complexes that carry out the light reactions5-13 (see also reviews14,15).
Freeze-fracture experiments of thylakoid membranes are typically carried out on preparations of chloroplasts or isolated thylakoid membranes (but see16,17), at the risk of any alteration in structural and/or supramolecular organization that may occur during the isolation procedure. Following fracture, replicas are prepared by evaporation of platinum/carbon (Pt/C), then by a thick layer of carbon (C), and finally digestion of the biological material18. Replicas are visualized by transmission electron microscopy (TEM). The traditional freeze-fracture-replica technique continues to serve as an important tool for studying the supramolecular organization of photosynthetic membranes and their adaption to different, e.g., light, conditions19-23.
In our recent study of the homoiochlorophyllous resurrection plant Craterostigma pumilum24, we aimed to investigate the changes in the supramolecular organization of thylakoid membranes, as well as in overall cellular organization, during dehydration and rehydration. The uniqueness of homoiochlorophyllous resurrection species is that they are able to survive conditions of desiccation in their vegetative tissues (leaves), while retaining their photosynthetic apparatus. Once water is available, these plants recover and resume photosynthetic activity within hours to a few days25. For this study, cryo-scanning EM (SEM) imaging of freeze-fractured leaf samples was combined with high-pressure freezing for sample cryo-immobilization. These procedures provide a means to visualize frozen-hydrated biological samples at a state close to their native state26. One main benefit is that samples are examined directly after freeze-fracture and coating with no successive steps. This is particularly relevant to the investigation of plants at different relative water contents (RWC), as their hydration state is maintained during preparation. However, one critical disadvantage is that frozen-hydrated samples may suffer from beam damage during imaging, especially when scanned at high magnifications, required for accurate measurement of the size of photosynthetic complexes. To overcome this, a method called &#39;double-layer coating&#39; (DLC)27,28 combined with specific cryo-SEM imaging conditions were utilized. These resulted in samples that are significantly less beam-sensitive and allowed for the elucidation of valuable information on photosynthetic protein supramolecular organization and other cellular constituents of the resurrection plant C. pumilum at high magnifications in situ.

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			<td height="21" style="height:21px;width:312px;">ethanol abs</td>
			<td style="width:359px;">Bio-Lab</td>
			<td style="width:119px;">052505</td>
			<td style="width:489px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">isopropanol</td>
			<td style="width:359px;">Bio-Lab</td>
			<td style="width:119px;">162605</td>
			<td style="width:489px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1-hexadecene</td>
			<td style="width:359px;">Sigma-Aldrich</td>
			<td style="width:119px;">H7009</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:312px;">0.1/0.2 platelets</td>
			<td style="width:359px;">Engineering Office M. Wohlwend GmbH, Switzerland</td>
			<td style="width:119px;">241</td>
			<td style="width:489px;">Platelets are of 3-mm diameter and 0.5-mm-thick (Type A) with 0.1/0.2-mm-deep cavities (of diamater 2 mm). Similar platelets can be obtained from Leica Microsystems.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">high-precision-grade tweezers</td>
			<td style="width:359px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">72706-01</td>
			<td style="width:489px;">Dumont (Switzerland) Durostar style #5 tweezers; Can be substituted with other high-precision tweezers.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:312px;">high-pressure freezing machine</td>
			<td style="width:359px;">Bal-Tec</td>
			<td style="width:119px;">HPM 010</td>
			<td style="width:489px;">High-pressure freezing alternatives: 1. HPF Compact 02, Wohlwend GmbH; 2. HPM 010, RMC Boeckeler; 3. EM PACT2, Leica Microsystems; 4. EM HPM 100, Leica Microsystems; 5. EM ICE, Leica Microsystems.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">freeze-fracture system</td>
			<td style="width:359px;">Leica Microsystems</td>
			<td style="width:119px;">EM BAF 060</td>
			<td style="width:489px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">cryo preparation loading stage</td>
			<td style="width:359px;">Leica Microsystems</td>
			<td style="width:119px;">16770228</td>
			<td style="width:489px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">specimen holder for univeral freeze fracturing</td>
			<td style="width:359px;">Leica Microsystems</td>
			<td style="width:119px;">16LZ04746VN</td>
			<td style="width:489px;">Clamp holder for specimen carriers of diameter 3 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">vacuum cryo-transfer shuttle</td>
			<td style="width:359px;">Leica Microsystems</td>
			<td style="width:119px;">EM VCT 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">scanning electron microscope</td>
			<td style="width:359px;">Zeiss</td>
			<td style="width:119px;">Ultra 055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">cryo SEM stage</td>
			<td style="width:359px;">Leica Microsystems</td>
			<td style="width:119px;">16770299905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">image acquisiton software</td>
			<td style="width:359px;">SmartSEM, Carl Zeiss Microscopy GmbH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">image analysis software</td>
			<td style="width:359px;">Fiji/Image J, National Institute of Health</td>
			<td style="width:119px;">http://fiji.sc/Fiji</td>
			<td></td>
		</tr>
	</tbody>
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studying the supramolecular organization of photosynthetic membranes within freeze-fractured leaf tissues by cryo-scanning electron microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Figure 1 shows cryo-SEM images of platelets containing high-pressure frozen, freeze-fractured Craterostigma pumilum leaf pieces. In some samples, large regions of fractured cells are obtained (Figure 1A). In others, the leaf piece stays tightly bound to the upper disc and is knocked off along with it (Figure 1B). However, even in the second case, some leaf tissue may remain attached to the knife grooves on the platelet (F...

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

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

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

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