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.
<ol>
	<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...

<ol>
	<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-Fracture+Studies+on+Barley+Plastid+Membranes+.6.+Location+of+the+P700-Chlorophyll+a-Protein-1.">Freeze-Fracture Studies on Barley Plastid Membranes .6. Location of the P700-Chlorophyll a-Protein-1.</a> Eur. J. Cell Biol. 31 (2), 305-314 (1983).</li><li>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=Reversible+Particle+Movements+Associated+with+Unstacking+and+Restacking+of+Chloroplast+Membranes.">Reversible Particle Movements Associated with Unstacking and Restacking of Chloroplast Membranes.</a> Invitro. J. Cell Biol. 71 (1), 136-158 (1976).</li><li>Miller, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Chloroplast+Membrane+Lacking+Photosystem-I+-+Changes+in+Unstacked+Membrane+Regions.">A Chloroplast Membrane Lacking Photosystem-I - Changes in Unstacked Membrane Regions.</a> Biochim. Biophys. Acta. 592 (1), 143-152 (1980).</li><li>Miller, K. R., Cushman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+Membrane+Lacking+Photosystem-II+-+Thylakoid+Stacking+in+the+Absence+of+the+Photosystem-II+Particle.">Chloroplast Membrane Lacking Photosystem-II - Thylakoid Stacking in the Absence of the Photosystem-II Particle.</a> Biochim. Biophys. Acta. 546 (3), 481-497 (1979).</li><li>Miller, K. 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=Analysis+of+Thylakoid+Outer+Surface+-+Coupling+Factor+Is+Limited+to+Unstacked+Membrane+Regions.">Analysis of Thylakoid Outer Surface - Coupling Factor Is Limited to Unstacked Membrane Regions.</a> J. Cell Biol. 68 (1), 30-47 (1976).</li><li>Simpson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-Fracture+Studies+on+Barley+Plastid+Membranes+.3.+Location+of+the+Light-Harvesting+Chlorophyll-Protein.">Freeze-Fracture Studies on Barley Plastid Membranes .3. Location of the Light-Harvesting Chlorophyll-Protein.</a> Carlsberg Res. Commun. 44 (5), 305-336 (1979).</li><li>Olive, J., Recouvreur, M., Girardbascou, J., Wollman, F. 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. A., Recouvreur, M., Bennoun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+Cytochrome+B6/F+Complex+.2.+Localization+of+the+Complex+in+the+Thylakoid+Membranes+from+Spinach+and+Chlamydomonas-Reinhardtii+by+Immunocytochemistry+and+Freeze-Fracture+Analysis+of+B6/F+Mutants.">Studies on the Cytochrome B6/F Complex .2. Localization of the Complex in the Thylakoid Membranes from Spinach and Chlamydomonas-Reinhardtii by Immunocytochemistry and Freeze-Fracture Analysis of B6/F Mutants.</a> Biochim. Biophys. Acta. 851 (2), 239-248 (1986).</li><li>Armond, P. A., Staehelin, L. A., Arntzen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Relationship+between+Light+Harvesting+Complex+and+Photosystem-1+and+Photosystem-2+in+Stacked+and+Unstacked+Chloroplast+Membranes.">Spatial Relationship between Light Harvesting Complex and Photosystem-1 and Photosystem-2 in Stacked and Unstacked Chloroplast Membranes.</a> J. Cell Biol. 70 (2), 400-418 (1976).</li><li>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=Chloroplast+structure:+from+chlorophyll+granules+to+supra-molecular+architecture+of+thylakoid+membranes.">Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes.</a> Photosynth. Res. 76 (1-3), 185-196 (2003).</li><li>Nevo, R., Charuvi, D., Tsabari, O., Reich, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition,+architecture+and+dynamics+of+the+photosynthetic+apparatus+in+higher+plants.">Composition, architecture and dynamics of the photosynthetic apparatus in higher plants.</a> Plant J. 70 (1), 157-176 (2012).</li><li>Platt, K. A., Oliver, M. J., Thomson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+and+Organelles+of+Dehydrated+Selaginella+and+Tortula+Retain+Their+Normal+Configuration+and+Structural+Integrity+-+Freeze-Fracture+Evidence.">Membranes and Organelles of Dehydrated Selaginella and Tortula Retain Their Normal Configuration and Structural Integrity - Freeze-Fracture Evidence.</a> Protoplasma. 178 (1-2), 57-65 (1994).</li><li>Platt-Aloia, K. A., Thomson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+the+use+of+intact+plant+tissues+in+freeze-fracture+electron+microscopy.">Advantages of the use of intact plant tissues in freeze-fracture electron microscopy.</a> J. Electron Microsc. Tech. 13 (4), 288-299 (1989).</li><li>Carson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+technical+elements+of+freeze-fracture/freeze-etch+in+biological+electron+microscopy.">Fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy.</a> J. Vis. Exp. (91), e51694 (2014).</li><li>Kirchhoff, H., 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=Low-light-induced+formation+of+semicrystalline+photosystem+II+arrays+in+higher+plant+chloroplasts.">Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts.</a> Biochemistry. 46 (39), 11169-11176 (2007).</li><li>Johnson, M. P., 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=Photoprotective+Energy+Dissipation+Involves+the+Reorganization+of+Photosystem+II+Light-Harvesting+Complexes+in+the+Grana+Membranes+of+Spinach+Chloroplasts.">Photoprotective Energy Dissipation Involves the Reorganization of Photosystem II Light-Harvesting Complexes in the Grana Membranes of Spinach Chloroplasts.</a> Plant Cell. 23 (4), 1468-1479 (2011).</li><li>Kirchhoff, H., Tremmel, I., Haase, W., Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+photosystem+II+organization+in+grana+thylakoid+membranes:+evidence+for+a+structured+arrangement.">Supramolecular photosystem II organization in grana thylakoid membranes: evidence for a structured arrangement.</a> Biochemistry. 43 (28), 9204-9213 (2004).</li><li>Belgio, E., Ungerer, P., Ruban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V+Light-harvesting+superstructures+of+green+plant+chloroplasts+lacking+photosystems.">V Light-harvesting superstructures of green plant chloroplasts lacking photosystems.</a> Plant Cell Environ. , (2015).</li><li>Goral, T. K., 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=Light-harvesting+antenna+composition+controls+the+macrostructure+and+dynamics+of+thylakoid+membranes+in+Arabidopsis.">Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis.</a> Plant J. 69 (2), 289-301 (2012).</li><li>Charuvi, 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=Photoprotection+Conferred+by+Changes+in+Photosynthetic+Protein+Levels+and+Organization+during+Dehydration+of+a+Homoiochlorophyllous+Resurrection+Plant.">Photoprotection Conferred by Changes in Photosynthetic Protein Levels and Organization during Dehydration of a Homoiochlorophyllous Resurrection Plant.</a> Plant Physiol. 167 (4), 1554-1565 (2015).</li><li>Farrant, J. M., Brandt, W., Lindsey, G. 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Biol. 184 (2), 355-360 (2013).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9 (7), 676-682 (2012).</li><li>Walther, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+freeze-fracturing+of+high-pressure+frozen+samples.">Recent progress in freeze-fracturing of high-pressure frozen samples.</a> J. 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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.

<table border="0" cellpadding="0" cellspacing="0" width="1278">
	<tbody>
		<tr height="21">
			<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>
</table>

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 ...

The overall goal of this technique is to study the supramolecular organization of photosynthetic membranes using cryo-scanning electron microscopy of freeze-fractured leaf tissue samples. This method can help answer key questions in cell biology, such as the morphological characterization of cells and organelles, the distribution of membrane-integral protein complexes, and localization of biominerals. The main advantage of this technique is that freeze-fracture is performed on leaf tissues, and so thylakoid membrane supramolecular organazation is investigated within the native organelle and cellular context.While this method is demonstrated here for a biological sample, it can also be applied to such as gels, polymers, and drug delivery systems. Generally, individuals new to these procedures should be aware that not all samples will be well-frozen or properly fractured. To carry out cryofixation of leaf tissues by high-pressure freezing, begin by using the corner of a razor blade to scratch the bottom of the 0.1-millimeter cavity of a 0.1-to-0.2-millimeter aluminum platelet.Prepare at least a dozen platelets for six samples. Then use absolute ethanol to wash the platelets, and keep them in a clean dish. After preparing the high-pressure freezing machine, fill the samples box with liquid nitrogen.With a razor blade, cut a small piece of leaf from the plant and use tweezers to place the tissue inside a microcentrifuge tube half filled with one hexadecane. To infiltrate the intercellular spaces of the leaf tissue, insert the tip of the infiltration device, prepared as described in the text protocol into the hole in the lid of the microcentrifuge tube, and pull the plunger to create vacuum. After about 30 seconds, release the plunger.Next, using tweezers, remove the leaf piece from the tube. Use the razor blade to cut a piece small enough to fit into a platelet. And if necessary, trim the leaf to a thickness of less than 0.2 millimeters.To prevent tissue collapse, leaves are infiltrated with liquid prior to high-pressure freezing. Sample thickness is kept below 200 microns in order to maximize the chance for obtaining a vitrified sample. Place a clean platelet with the scratch side up into the freezing machine's holder.And then place the cut piece of leaf into the platelet. Use one hexadecane to fill the remaining space of the platelet before using another platelet with the scratches facing the leaf to close the sandwich. Close and tighten the holder.Freeze the sample by pressing the jet button. Then, quickly transfer the holder into liquid nitrogen. Use precool tweezers to remove the sandwich from the holder, taking care not to fracture it.There are two critical steps in the procedure:freeze-fracture, which is a random process and its success can only be assessed in the cryo-SEM'and double-layer coating, which is required for obtaining high-magnification images with minimal beam damage. While preparing and programming the freeze-fracture machine according to the manufacturer's instructions, place the platinum carbon, or Pt/C gun, at 45 degrees, and the carbon gun at 90 degrees. Cool the freeze-fracture system stage to minus 140 or minus 160 degrees Celsius.Then attach the vacuum cryo transfer shuttle to the machine and use liquid nitrogen to fill the shuttle chamber. Insert a freeze-fracturing specimen holder into the loading station, and with liquid nitrogen flood the loading station chamber. Next, using high-precision-grade tweezers, load a frozen sandwich onto the holder and tighten it in place.Then flip the holder over to check that the sandwich is secured in place, and repeat for a second sandwich. Detach the cooled shuttle from the freeze-fracture machine and connect it to the loading station. Then open the shuttle valve, introduce the retracting arm into the holder, lock it in place, and reintroduce the arm with the holder into the shuttle.After reattaching the shuttle to the freeze-fracture machine introduce the holder into the chamber, using the retracting arm. Now align the freeze-fracture microtome knife to height such that it will knock off the top platelets of the sandwiches. With the microtome knife, quickly break the sandwiches.And then immediately raise up the knife to prevent contamination of the samples by debris clinging to the knife. Position the cooled shutter above the newly-exposed plane to shield the samples from possible contamination by water molecules in the chamber. Next, to coat the samples with platinum, press Setup to show Layer 1 on the screen.Then turn on the Pt/C gun by pressing the GUN 1 button followed by the ON button. Examine the evaporation rate, and once it reaches 0.02 to 0.04 nanometers per second, press the Measure button to monitor the thickness of the deposited layer while simultaneously exposing the samples. To coat with carbon, select GUN 2 followed by the ON button.Once the evaporation rate is approximately 0.1 nanometers per second, press the Measure button and at the same time move the shutter to expose the samples. When the coating is complete, warm the stage to minus 120 degrees Celsius. To prepare the scanning electron microscope for cryo work, from the main menu select Stage, Stage Initialize, and confirm.Under the Vacuum tab, click the Vent button to vent the microscope chamber. Then select Tools, Goto Panel, and open the Stage Points List panel. Select the CRYO EXCHANGE position to move the stage to the proper coordinates for accepting the sample holder.While wearing a clean pair of gloves, open the specimen chamber and insert the cryostage onto the main stage of the microscope. Close the chamber, and under the Vacuum tab, click the Pump button. Next, cool the microscope by filling the microscope duor with liquid nitrogen.Once the temperature of the stage drops below minus 120 degrees Celsius, activate the heat function on the cryo transfer unit to warm the stage to minus 120 degrees Celsius. Now attach the cryo transfer shuttle to the microscope and transfer the sample holder using the retracting arm into the microscope cryo stage. Using the joystick, carefully move the specimen holder close to the objective lens.In the Apertures tab, select the 10-micron aperture. Then, in the Gun tab, double-click the EHT Target field, enter 10 kilovolts for the EHT target, and click OK.Then, to turn on accelerating voltage, click the bottom EHT tab and select EHT On.In the Detectors tab, select InLens from the drop-down list. Then click on the Scanning tab and choose a scan speed equal to one, which is a fast scan to minimize beam damage, and a store resolution of 1024 by 768 pixels.In the Noise Reduction dropdown list, select Line Average, and adjust the value of n to 20 to 30 lines for searching. Then, by clicking the Control and Tab keys on the keyboard, use the centerpoint function to move the sample to areas of interest while simultaneously magnifying to identify regions with fractured cells. After identifying regions with fractured cells, scan the sample while magnifying to find a fractured chloroplast.Then change the line average n value to greater than 100, for noise reduction. In the Scanning tab, press Freeze to stop the scanning. By pressing Control Shift Tab on the keyboard, the center-feature function is used to zoom into interesting chloroplast-fracture faces;and high-magnification images are acquired using the same parameters as before.Finally, press the Save TIFF button to save the image. Carry out image analysis according to the text protocol. This figure shows a cryo-SEM image of a platelet containing a high-pressure frozen freeze-fractured Craterostigma Pumilum leaf piece with a large region of fractured cells.In this example, the leaf piece is missing;but some leaf tissue remained, attached to the knife grooves on the platelet. After a successful fracture is found, low-magnification images of cells are acquired to identify regions of interest, such as fractured chloroplasts. Images of single-fractured chloroplasts are acquired to identify thylakoid membrane fracture faces.Shown in this example is a high-magnification image of thylakoid membranes in C.Pumilum leaves. The four different fracture faces can be distinguished. The exoplasmic fracture faces of stacked and unstacked membranes, EFs and EFu respectively, and the protoplasmic faces of stacked and unstacked membranes, PFs and PFu respectively.Photosystem II, or PS2, is located in both the EFs stacked grana membrane regions and EFu stromalamellar membrane regions. When hydrated, PS2 density is approximately three times higher in the EFs than in the EFu, which differentiates the two faces. During dehydration of C.Pumilum, the density of PS2 in the EFs gradually decreases, reaching roughly half of its density at hydrated conditions.Notably, upon further dehydration to five to 10%relative water content, PS2 complexes organize into rows and arrays. Development of the freeze-fracture replica technique paved the way for studying the size of membrane protein complexes and their arrangement within biological membranes. The combination of freeze-fracture with high-pressure freezing in cryo-SEM allows investigating various kinds of samples at several size scales, from the tissue and cellular levels down to the macromolecular level at a near-native state.In addition to the technique shown here, methods such as Energy-Dispersive x-ray Spectroscopy, EDS, can also be applied in the cryo-SEM;for instance, for alkalizing biominerals.

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11.9K ビュー.  Weizmann Institute of Science. The overall goal of this technique is to study the supramolecular organization of photosynthetic membranes using cryo-scanning electron microscopy of freeze-fractured leaf tissue samples. This method can help answer key questions in cell biology, such as the morphological characterization of cells and organelles, the distribution of membrane-integral protein complexes, and localization of biominerals. The main advantage of this technique is that freeze-fracture is performed on leaf tissues, a...