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

Summary

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.

Abstract

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 (>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.

Introduction

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 'thylakoid' 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 app....

Protocol

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.

1. 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 platelets (6 samples). Take caution not to create knife marks on the circumference of the disc.
   1. After scratching, wash discs in absolute et....

Results

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.......

Discussion

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 state^26,32. The benefit of using leaf tissues is that one can obtain information on the overall cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol abs	Bio-Lab	052505
Isopropanol	Bio-Lab	162605
1-hexadecene	Sigma-Aldrich	H7009
0.1/0.2 Platelets	Engineering Office M. Wohlwend GmbH, Switzerland	241	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.
High-precision-grade tweezers	Electron Microscopy Sciences	72706-01	Dumont (Switzerland) Durostar style #5 tweezers; Can be substituted with other high-precision tweezers.
High-pressure freezing machine	Bal-Tec	HPM 010	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.
Freeze-fracture system	Leica Microsystems	EM BAF 060
Cryo preparation loading stage	Leica Microsystems	16770228
Specimen holder for univeral freeze fracturing	Leica Microsystems	16LZ04746VN	Clamp holder for specimen carriers of diameter 3 mm
Vacuum cryo-transfer shuttle	Leica Microsystems	EM VCT 100
Scanning electron microscope	Zeiss	Ultra 055
Cryo SEM stage	Leica Microsystems	16770299905
Image acquisiton software	SmartSEM, Carl Zeiss Microscopy GmbH
Image analysis software	Fiji/Image J, National Institute of Health	http://fiji.sc/Fiji