EM and EM-based techniques

Electron microscopy (EM) is the most common used technique to observe the nuclear structure. Electron microscope uses a beam of accelerated electrons as a source of illumination providing a high resolution, such as conventional transmission electron microscopy (TEM) with ~1 nm resolution. Since long, using TEM, one can observe thin sections of nuclei from chemically fixed or cryofixed cells. For conventional fixation methods, the cells are fixed by chemicals which can alter the nuclear morphology (Figure 23A). Cryo- fixation is a physical fixation technique that reduces fixation artifact and therefore preserve the nuclear ultrastructure (Figure 23B) (Trumtel et al., 2000). After cryo-fixation, following cryo-substitution and resin embedding to prepare the ultra-thin sections are also source of artifacts. Morover, the analysis of the ultrathin sections (50-100 nm) only allows the measurement of nuclear shape and size in 2D. To estimate the nuclear size in 3D from the 2D results, one has to do statistical analysis of many sections which is technical challenging. In addition, this method is generally biased due to the random sectioning orientation.

Figure 23. Nuclear morphology of yeast L1489 prepared by cryo-fixation and chemical fixation techniques, respectively. From (Trumtel et al., 2000).

A. Cryo-fixation better preserves the nuclear structure, even the sub-structures of the nucleus (the nucleolar structure).

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B. Chemical fixation alters the nuclear structure. While the sub-structures of the nucleolus, FC, DFC and GC, still can be observed, the nuclear envelope appears clearly “broken” and the general shape of the nuclear section is modified. Scale bars, 300 nm.

One way to detect the nuclear shape and size in 3D based on the EM technique is to analyze serial sections of an entire nucleus (Winey et al., 1997; Yamaguchi et al., 2011). Ultra-thin serial sections (60 nm thick) of cryofixed cells are harvested and analysed in order. For each section, the NE position is registred in order to reconstruct the 3D NE (Figure 24) (Winey et al., 1997). Winey et al. used this method to detect the nuclear shape and size in different cell cycle phases of budding yeast cells. In G1 and S phase, the nucleus is spherical and the average size increases ~21%. In early mitosis, the nuclear size is almost twice bigger than in G1 phase. In mitosis, the nucleus adopts an hour-glass shape. Although the resolution of EM is very high, however, for each single nucleus, there is no guarantee that all the serial sections are intact; this technique is complex and time-consuming. For each cell cycle stage, Winey et

al. just acquired ~10 nuclei, and it is difficult to acquire the nucleus in mitosis because of the

random sectioning orientation.

Figure 24. 3D reconstruction of the S. cerevisiae nuclear envelope analyzed by TEM. From (Winey et al., 1997).

A. Micrography of an ultra-thin section (from a serie) of a yeast nucleus analyzed by TEM.. Scale bar, 0.5 μm.

B. Detection of the positions of NPC and NE in each section, red circles represent the NPCs, green curve represents the NE.

C. Combination of all the NE contours and NPCs positions from serial sections to reconstruct the 3D NE.

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Figure 25. 3D reconstruction of the yeast cells by SBF-SEM technology. From (Miyazaki et al., 2014).

A. Acquisition of the images of the serial block-faces (XY plane, XZ plane and YZ plane). Scale bar, 5 μm.

B. Reconstruction of the 3D morphology of the yeast cell based on these block-face images. The outer semitransparent surface is the cell surface. The red volume represents the nucleus. The blue volume represents the vacuoles. The mitochondria are colored green. Scale bar, 5 μm.

C. Reconstruction of the 3D morphology of several yeast cells simultaneously. Scale bar, 5 μm.

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Miyazaki et al. proposed one technique, serial block-face scanning electron microscopy (SBF-SEM), to study the fine structures and morphological changes at nanometer scales in yeast cells. The sample is typically fixed with aldehyde, staining with heavy metals and then embedding in a resin. The surface of the block of resin-embedded sample was imaged by detection of back-scattered electrons. Following acquisition, the ultramicrotome which was mounted inside the vacuum chamber of a SEM was used to cut thin sections (typically less than 30 nm) from the face of the block. After the section was cut, the sample block was raised back to the focal plane and imaged again (Figure 25A). These serial block-face images allow to reconstruct the 3D morphology of the cells (Figure 25B) (Miyazaki et al., 2014). This technique also allows to visualize the organelle structures in cells, such as mitochondria, vacuoles, the ER and the nucleus, which can help us to study the cell processes.

SBF-SEM is a promising tool to detect several cells simultaneously. This is because SBF- SEM allows the examination of large volumes, such as 21×21×50 μm3, so one can segment several cells from a single reconstructed volume (Figure 25C). However, the sample number is still not enough to get robust statistical data. Furthermore, the preparation of the samples is still based on the fixation and section techniques, which maybe altere geometry of the cells compared to living yeast cells.

In conclusion, EM and EM-based techniques require fixation and sectioning steps of the samples which may influence the nuclear shape. The integrity of the serial sections which is essential for reconstruction of the 3D morphology of a nucleus is not guaranteed. Moreoevr, these approaches are extremely long, tedious and time-consuming which prevent to acquire enough data to perform robust statistical analysis. Finally, these techniques are not compatible with in vivo acquirement.