Electron tomography of cells

Abstract

The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or 'tomogram'. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.

Fig. 1.

Timeline of key developments. Key technologies and applications of EM are shown. The gray gradients emphasize that each of these development took many years.

Fig. 2.

Principle of electron tomography. (a) A tilt series of projection images is acquired from the object as it is incrementally tilted. (b) The tomogram is generated by back-projecting the aligned projection images. Ribosome coordinates (E. coli 70s) are from PDB entries 2AVY and 2AW4 (Schuwirth et al., 2005).

Fig. 3.

Correlated light and electron microscopy. (a) Correlation of mCherry-tagged McpA (inset, red) in a light micrograph of a C. crescentus cell (inset, dotted outline) and of the same cell in an electron cryotomogram. The chemoreceptor array is indicated by an arrow. (b) Low-magnification cryo-EM image showing the positions of individual segmented microtubules (yellow lines), which match those identified by the α-tubulin-GFP fluorescence cryo-light microscopy image of the same cell (upper right panel). (c) Cryotomographic slice of the region boxed in (b) showing microtubules (M), actin filaments (A), and putative ribosomes (asterisks) in a PtK2 cell. Reproduced with permissions from (a) Briegel et al. (2008) and (b and c) (Schwartz et al., 2007).

Fig. 4.

Electron tomography of vesicular trafficking systems. (a) Tomographic slice of a synapse showing a synaptic body (dense round body ~ 200 nm wide), Ω-shaped presynaptic membrane invagination (long arrow), and presynaptic density (short arrow). (b) Tomographic slice and model (colored) of part of a Golgi Body from a normal rat kidney cell. Different colors represent different cisternae. ER-Golgi intermediate compartment – yellow; trans-most cisterna – red. (c) Rendering of the model in 3-D. Reproduced with permissions from (a) Lenzi et al. (1999) and (b and c) Ladinsky et al. (1999).

Fig. 5.

Electron tomography of spindle microtubules. (a) Nuclear (upper panels) and cytoplasmic (lower panels) microtubules from tomograms of S. cerevisiae cells. (b) Low-magnification EM image of a kinetochore (boxed) from a PtK1 cell. Chromosomes are the lark, densely stained objects. (c) Tomographic slice of the region boxed in (b), showing the curved morphologies of two kinetochore microtubule plus ends. (d) Gallery showing that kinetochore plus ends exhibit both blunt/straight and curved (ram’s horn) morphologies. Reproduced with permissions from (a) O’Toole et al. (1999) and (b – d) Vandenbeldt et al. (2006).

Fig. 6.

Electron cryotomography of eukaryotic cells. (a) Surface rendering showing actin (red), putative ribosomes (green), and membranes (blue) at the periphery of a frozen-hydrated Dictyostelium discoideum cell. (b) Cryotomographic slices of an intact Dictyostelium nucleus showing top (left) and side (right) views of the nuclear pore complexes (NPC, arrows). Patches of ribosome-studded rough ER are indicated by arrowheads. (c) Subtomographic average of the NPC. (d) Probability density (orange) of gold-labeled classical import cargoes. Reproduced with permissions from (a) Medalia et al. (2002), (b) Beck et al. (2004), and (c and d) Beck et al. (2007).

Fig. 7.

Electron cryotomography of vitreous tissue sections. (a) Cryo-EM image of a cryosectioned human skin biopsy showing desmosomes (D) and filament networks (IF). (b) Isosurface rendering of a cryotomogram from another region of skin showing desmosomes (gold/brown), the nucleus (blue), nuclear pores (red), and a putative microtubule (green). (c) Cryotomographic slice of a desmosome showing extracellular (EC) and intracellular (IC) spaces. (d) Subtomographic average of C-cadherin molecules (white isosurfaces) with crystal structures docked. Reproduced with permission from Al-Amoudi et al. (2007).

Fig. 8.

Transcytosis followed in tissues. (a) Tomographic slice of the lateral intercellular space (LIS) between two neonatal rat jejunal cells. Gold-labeled Fc (Au-Fc) molecules can be found in both the LIS (red arrows) and irregular vesicles (white arrows). (b) 3-D segmentation of the region in (a) showing membranes and vesicles in green and blue, and Au-Fc in gold. Reproduced with permission from He et al. (2008).

Fig. 9.

Insights into bacterial ultrastructure. (a) Cryotomographic slice of a Caulobacter crescentus cell showing the inner curvature filaments (white dashed box). Other features include the S-layer (SL), outer membrane (OM), peptidoglycan layer (PG), inner membrane (IM), stalk (St), putative ribosomes (Rib), putative poly-β-hydroxybutyrate granule (Phb), and gold fiducial (GF). (b) Isosurface rendering of a Treponema primitia flagellar motor subtomographic average. (c) Cutaway of (b), with components labeled: peptidoglycan-binding collar (P), stator (S), rotor (R), cytoplasmic ring (C), putative export structure (E), and inner membrane (IM). (d) Cartoon of the motor’s organization based on the cryotomography data. Reproduced with permissions from Briegel and Dias et al. (2006) and Murphy et al. (2006).

Fig. 10.

Virus-cell interactions. (a) Tomographic slice (left) and surface rendering (right) of HIV-1 bound to a T cell. Structures are colored as: viral membrane (purple), core (yellow), cell membrane (blue), and “entry claw” contacts (red). (b) Cryotomographic slice of a Sulfolobus solfataricus cell, infected with Sulfolobus turreted icosahedral virus (STIV). Virus-induced pyramids appeared with electron-dense bodies (black arrow) or without supermolecular complexes (white arrow). (c) Cryotomographic slices of partially assembled (p), empty (e), and filled (f) STIV. (d) Central slices through the subtomographic averages of full (left) and emptied (right) ε15 bacteriophages bound to Salmonella hosts. Reproduced with permissions from Sougrat et al. (2007), Fu et al. (2010), and Chang et al. (2010).

Fig. 11.

Zernike phase-contrast. Cryo-EM of GroEL with (a) defocus phase contrast and (b) Zernike phase contrast generated by a thin amorphous-carbon phase plate. Reproduced with permission from Danev et al. (2008).