EM tomography (ET) enables 3D reconstructions of parts of cells or of whole
cells, in the case of bacteria, at 4–6 nm resolution in 3D and thus has been a popular
method for studying structure-function relationships among organelles [14, 15].
This method involves the use of a TEM to collect tilted views of a thick (200–
400 nm) section over a wide angular range (typically ±60 or 70°). The tilted views
are then aligned, and a reconstruction is computed. ET is best suited for reconstructing objects that change significantly within a small volume, such as the
twisting contours of intracellular membranes or complex networks of cytoskeletal
elements (e.g., the mitotic spindle). Cryo-electron tomography (cryoET) is a specialized form of ET in which frozen-hydrated material is imaged, allowing material
to be imaged in a near-native state [16]. In CryoET, the specimen suffers from
damage during exposure to the electron beam, and this limits imaging large areas
and volumes. For this reason, this chapter focuses on the large area and volume
reconstruction from plastic-embedded sections. A separate chapter describing the
utility of using cryoET is included in Chap. 3.
For plastic section tomography, larger volumes have been generated by stacking
tomograms of serial thick (250–400 nm) sections along the Z axis (ssET), a logical
extension of the more established serial section microscopy with thin sections [17,
18]. In this way, one can progress through the sample in successive 3D slabs to
generate what amounts to a column of tomographic data. In the first study that used
tools in IMOD for ssET, serial sections of *250 nm thickness were imaged with
1.5° of angular change between tilted views and digitized at a pixel size of 2.3 nm.
This study achieved a resolution of *7 nm revealing the complex membranes of
the Golgi complex in considerable detail (Fig. 4.1a, [18]). The first reconstruction
of an entire mammalian cell was done with a murine pancreatic b cell (Fig. 4.1b,
[19]). By reducing the magnification to a nominal *4700Â, such that the entire
cell cross section could be visualized in a single field of view, and capturing
tomograms through *40 serial sections, an entire mammalian cell was imaged in
3D with *5 nm voxels, providing a resolution of 10–15 nm.
Areas larger than the frame size of the CCD camera at a given magnification can
be captured by montaging. The use of montaging in the plane of each section at
higher magnification, combined with serial section tomography was used to
reconstruct whole cells from the yeast, Schizosaccharomyces pombe, allowing the
details of the interphase microtubule cytoskeleton to be visualized with unprecedented resolution. Details such as the morphology of microtubule ends could then
be analyzed in the natural cellular context (Fig. 4.1c, [20]). To increase the area of
the reconstructed volume even further, we have developed methods for stitching
together laterally adjacent tomograms, which we refer to as supermontages
(Fig. 4.1d, [21]. Stacking supermontage tomograms allows the reconstruction of
significant cell volumes, such as the mitotic spindle of C. elegans (Fig. 4.1d). An
alternative approach using TxBR montage reconstruction has also been described
[22].
In this chapter, we describe the use of SerialEM and IMOD to enable reconstructions of relatively large volumes of sectioned material with an emphasis on
recent advances in automation for both image acquisition and tomographic
4 Large-Scale Electron Tomography of Cells …
97
cells, in the case of bacteria, at 4–6 nm resolution in 3D and thus has been a popular
method for studying structure-function relationships among organelles [14, 15].
This method involves the use of a TEM to collect tilted views of a thick (200–
400 nm) section over a wide angular range (typically ±60 or 70°). The tilted views
are then aligned, and a reconstruction is computed. ET is best suited for reconstructing objects that change significantly within a small volume, such as the
twisting contours of intracellular membranes or complex networks of cytoskeletal
elements (e.g., the mitotic spindle). Cryo-electron tomography (cryoET) is a specialized form of ET in which frozen-hydrated material is imaged, allowing material
to be imaged in a near-native state [16]. In CryoET, the specimen suffers from
damage during exposure to the electron beam, and this limits imaging large areas
and volumes. For this reason, this chapter focuses on the large area and volume
reconstruction from plastic-embedded sections. A separate chapter describing the
utility of using cryoET is included in Chap. 3.
For plastic section tomography, larger volumes have been generated by stacking
tomograms of serial thick (250–400 nm) sections along the Z axis (ssET), a logical
extension of the more established serial section microscopy with thin sections [17,
18]. In this way, one can progress through the sample in successive 3D slabs to
generate what amounts to a column of tomographic data. In the first study that used
tools in IMOD for ssET, serial sections of *250 nm thickness were imaged with
1.5° of angular change between tilted views and digitized at a pixel size of 2.3 nm.
This study achieved a resolution of *7 nm revealing the complex membranes of
the Golgi complex in considerable detail (Fig. 4.1a, [18]). The first reconstruction
of an entire mammalian cell was done with a murine pancreatic b cell (Fig. 4.1b,
[19]). By reducing the magnification to a nominal *4700Â, such that the entire
cell cross section could be visualized in a single field of view, and capturing
tomograms through *40 serial sections, an entire mammalian cell was imaged in
3D with *5 nm voxels, providing a resolution of 10–15 nm.
Areas larger than the frame size of the CCD camera at a given magnification can
be captured by montaging. The use of montaging in the plane of each section at
higher magnification, combined with serial section tomography was used to
reconstruct whole cells from the yeast, Schizosaccharomyces pombe, allowing the
details of the interphase microtubule cytoskeleton to be visualized with unprecedented resolution. Details such as the morphology of microtubule ends could then
be analyzed in the natural cellular context (Fig. 4.1c, [20]). To increase the area of
the reconstructed volume even further, we have developed methods for stitching
together laterally adjacent tomograms, which we refer to as supermontages
(Fig. 4.1d, [21]. Stacking supermontage tomograms allows the reconstruction of
significant cell volumes, such as the mitotic spindle of C. elegans (Fig. 4.1d). An
alternative approach using TxBR montage reconstruction has also been described
[22].
In this chapter, we describe the use of SerialEM and IMOD to enable reconstructions of relatively large volumes of sectioned material with an emphasis on
recent advances in automation for both image acquisition and tomographic
4 Large-Scale Electron Tomography of Cells …
97