Introduction

With the 2.91 billion base pairs of the human genome mapped [1-3], one of the main challenges facing science is to understand the functioning of more than 26,000 encoded proteins. For the overwhelming majority of proteins it is not well understood why a certain amino acid sequence leads to a specific tertiary structure into which the protein folds [4]. Only for very small molecules it is possible to numerically calculate their folding in a reliable manner. Our true mastery of self-assembly is therefore limited to relatively simple systems [5-7]. Many questions remain open concerning the highly complex organization of the proteins into functional cells. The limited comprehension of protein and cell function is mainly due to a lack of detailed structural information [4,8]. To date only about 90 unique structures of membrane proteins have been resolved [4]. Moreover, the organization of proteins in cells has only been accessible so far by techniques that do not combine high spatial resolution with imaging in their native environment, or the imaging of dynamical behavior.

Ideally, one would like to have access to an imaging technique providing the eight requirements listed in Table 13.1. Only such a technique allows a direct, in vivo, study of the function of the molecular machinery. Of secondary importance, but in many cases a limiting factor is obviously the cost of the apparatus and its operation. Figure 13.1 schematically presents the fulfillment of the eight main requirements versus the resolution of the technique. A trend exists in which better resolution can be achieved only at the cost of less direct imaging of the functioning of the cell, subunit, or protein.

Figure 13.1 illustrates that a clear need and drive exists to push existing techniques and develop new techniques that provide high-resolution imaging with as close to in vivo capabilities as possible. At a resolution below 1 nm already much can be gained when only four or five requirements are met, whereas in the region of a few to several tens of nanometers resolution seven requirements can be met. Electron microscopy (EM) techniques based on averaging over many images of a single type of particle continue to push the limit on the high-resolution side [9], whereas on the tens of nanometers side confocal laser microscopy is gaining ground [10].

Recent instrumental developments have enabled drastic improvements in the resolution of STEM using aberration correction [11]. An additional and previously unanticipated advantage of aberration correction is the greatly improved depth sensitivity that has led to the reconstruction of a 3D image from a focal series [12,13]. In this chapter we will discuss the potential of aberration-corrected 3D STEM to

TABLE 13.1 Requirements for the Imaging of Biological Function in Addition to High Resolution

Number Requirement

1 3D imaging

2 In natural liquid environment, i.e., not frozen

3 Single particles, i.e., no crystals

4 The whole assembly comprising, for example, many proteins reacting together, or a whole protein complex and not only small subunits

5 Time-resolved

6 Intracellular, not only surface

7 Reproducibility

8 Fast imaging

Resolution/nm

FIGURE 13.1 Number of fulfilled requirements for the imaging of the functioning cell, or subunit in vivo versus the resolution for various imaging techniques. EM tomography means electron microscopy tomography. The figure is meant as guide for the discussion and by no means claims absolute limits of a certain technique. The ellipse with the question mark indicates the specifications of the ideal technique.

Resolution/nm

FIGURE 13.1 Number of fulfilled requirements for the imaging of the functioning cell, or subunit in vivo versus the resolution for various imaging techniques. EM tomography means electron microscopy tomography. The figure is meant as guide for the discussion and by no means claims absolute limits of a certain technique. The ellipse with the question mark indicates the specifications of the ideal technique.

provide major improvements in the imaging capabilities for biological samples. First, we will give a brief overview of the different high-resolution 3D techniques and then we will introduce the reader to some of the history of EM, STEM, and aberration correction. In Section 13.3.6 the concept of 3D STEM will be described. Sections 13.4-13.5 will evaluate the potential of 3D STEM for high-resolution 3D imaging of stained biological samples.

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