Conical tomography II: A method for the study of cellular organelles in thin sections

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Abstract

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55° tilt and at 5° rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the “unit membrane pattern” along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the “unit membrane” pattern, we estimated resolutions of ∼4 nm. To illustrate the prospective advantages of conical tomography, we segmented “coated” vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein “coating” extending into the cytoplasm. The “coated” vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of ∼4 nm.

Introduction

We have used the general method of electron tomography in order to determine the 3D structure of macromolecular assemblies and organelles in their cellular environments. By imaging the same organelle or assembly in different directions, we collect a series of images referred to as the “tilt series.” Processing this “tilt series” allows the calculation of 3D maps that describe these assemblies and organelles at resolutions that are at least one order of magnitude higher than optical methods (Hoppe and Hegerl, 1980). The accuracy with which the 3D map represents the structure of organelles and assemblies depends on how much of the original volume is sampled in the tilt series. Due to the technical limitations of the electron microscope, all reconstructions by tomography contain regions devoid of information, aptly named “missing regions” (Turner and Valdrè, 1992). The magnitude and position of these regions depend on several factors including the tilt angle and the geometry used to collect the series (single-axis, double-axis, and conical tilt). The geometry used to collect the tilt series directly affects the accuracy with which organelles oriented parallel to the tilt axis are resolved in the XY plane (the “anisotropic” resolution). This anisotropy is largest in single-tilt, diminished in double-tilt (Penczek et al., 1995), and eliminated in conical tilt (Lanzavecchia et al., 2005). In all three geometries, limited tilt angles (<45°) induce severe elongation along the Z-axis.

In a previous study, we used conical series to reconstruct freeze-fracture replicas (∼1.5 nm in thickness) of liposomes reconstituted with the lens water channel aquaporin-0 (AQP0). Calculations using Fourier shell correlation (FSC) indicated a resolution of 2–3 nm, permitting accurate estimates of the size, shape, and volume of AQP0 tetramers (Lanzavecchia et al., 2005). In this study, we applied the same general method of conical tomography to reconstruct thin sections of the rat somato-sensory cortex, which are ∼30 times thicker and more sensitive to radiation damage than the metal replicas. To improve the “global” resolution of the reconstructions, we used a process involving projection matching to refine the reconstructions. This technique resulted in maps with a resolution of ∼4 nm—high enough to reveal the bilayer organization of phospholipids in biological membranes.

Section snippets

Thin sectioning

All methods have been published previously (Zampighi and Fisher, 1997). Briefly, three Sprague–Dawley adult rats 90–120 days of age were sacrificed by overdose of Na pentobarbital (100 mg/kg body weight) and perfused transcardially with 300–500 ml of 150 mM NaCl. Primary fixation was performed by perfusion of ∼1 liter of 4% paraformaldehyde, 3% glutaraldehyde in 0.2 M Na cacodylate, pH 7.3. The brains were removed and immersed in the same fixative overnight. One hundred to 150 μm slices were cut

Technical considerations

Collection of conical series was a straightforward process thanks to the simplicity in operating the rotating/tilting stage and the advantages of imaging with a CCD camera. In aligning the projections, the selection of the common center was greatly simplified by using gold particles as fiduciary markers attached to the thin sections’ surface. Since preliminary values for the Euler angles were obtained experimentally (α = 5°, β = 55°, and γ = 0°), the elliptical paths of other gold particles present

Discussion

This paper demonstrates that conical electron tomography allows 3D reconstruction of thin sections of plastic embedded cortical tissue, which were thicker (∼50 nm) and more sensitive to radiation damage than metal replicas (Lanzavecchia et al., 2005). The practical and computational solutions advanced in this paper permitted the collection of up to two complete tilt series in an approximately a single session. Furthermore the entire process, from the collection to the fully reconstructed map,

Acknowledgments

This work was supported by Grants EY-04410, 1R21 DK60846 (G.Z.), DK44602 (E.M.W.) and FIRST 2001 (S.L. and F.C.) and the Research To Prevent Blindness Foundation (G.Z.).

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