52
3 Principle and Practice of Three-Dimensional Transmission …
In order to construct the three-dimensional (3D) images, we have to make use of
computerized tomography, which is a technique for 3D image by displaying crosssectional images through a solid object using ultrasound, X-ray or, in the case of TEM,
electron beam, followed by 3D image construction procedure using a computer. In
practice, the thin specimen is mounted on a specific sample holder for 3D-TEM,
which is capable to be rotated. The sample holder is successively tilted, and at each
tilting, TEM measurement is conducted to obtain a TEM image (a slice) and it is
sent to and stored in the computer. The slice images are subjected to tomographic
reconstruction of a 3D image of the specimen.
3.2.2 Tomography Applied to TEM
In our study, a Tecnai G2F20 TEM (FEI Co.) setup at NISSAN ARC, LTD was
used to measure unstained specimens at an accelerating voltage of 200 kV. The
specimens showed sufficiently good contrast between inorganic fillers and organic
rubbers without any staining: The brightness in the image is divided into 256 levels
from white to black. It was lucky for us that the electron staining [9–12] was not
required. The staining quite often brought about the structural modification of the
sample [2, 6, 8, 13]. However, the images here are the ones after the removal of
organic zinc compounds from the specimen by using organic solvents in order to
enhance the contrast, as described later in Sect. 4.2.1. After checking through all the
obtained images, the intensity level of 190–255 was assigned to inorganic particles.
The transition was sharp enough that it was not necessary to consider intermediate
layers between inorganics and rubber, which implies that TEM did not recognize the
interface layer of rubber (bound rubber or immobilized rubber layer) on the inorganic
surface.
The procedure for 3D-TEM measurements is outlined in Fig. 3.2 [8]. It is divided
into two steps, with a computer playing a major role in each step. Specimens of
approximately 200 nm thickness are prepared using a cryostat microtome. For the
fiducial marker method [14], colloidal gold particles of 10 nm diameter are spread
on the specimens before mounting it on a holder. While tilting the sample grid in
2° steps within the angle range from −70° to +70°, it is irradiated with the electron
beam to capture TEM images. The images are not simply 2D image slices, but 2D
projections of the mass–density distribution of the specimens. During the tilts, the
positions of the tilted images are aligned together with the axis of rotation being
calibrated by the fiducial marker and a computerized positioning of the relationship
between the images. The calibration due to the different thicknesses with the tilt
angles was automatically carried out during the measurement. The procedure up to
this point constitutes the first step.
The second step consists of computerized tomography, which is in principle the
same as a medical X-ray CT scan. The sequence of tilted images obtained is subject
to the Radon transform followed by the inverse Radon transform to reconstruct the
3D image in the real space (Radon transform is a kind of Fourier transform on
3 Principle and Practice of Three-Dimensional Transmission …
In order to construct the three-dimensional (3D) images, we have to make use of
computerized tomography, which is a technique for 3D image by displaying crosssectional images through a solid object using ultrasound, X-ray or, in the case of TEM,
electron beam, followed by 3D image construction procedure using a computer. In
practice, the thin specimen is mounted on a specific sample holder for 3D-TEM,
which is capable to be rotated. The sample holder is successively tilted, and at each
tilting, TEM measurement is conducted to obtain a TEM image (a slice) and it is
sent to and stored in the computer. The slice images are subjected to tomographic
reconstruction of a 3D image of the specimen.
3.2.2 Tomography Applied to TEM
In our study, a Tecnai G2F20 TEM (FEI Co.) setup at NISSAN ARC, LTD was
used to measure unstained specimens at an accelerating voltage of 200 kV. The
specimens showed sufficiently good contrast between inorganic fillers and organic
rubbers without any staining: The brightness in the image is divided into 256 levels
from white to black. It was lucky for us that the electron staining [9–12] was not
required. The staining quite often brought about the structural modification of the
sample [2, 6, 8, 13]. However, the images here are the ones after the removal of
organic zinc compounds from the specimen by using organic solvents in order to
enhance the contrast, as described later in Sect. 4.2.1. After checking through all the
obtained images, the intensity level of 190–255 was assigned to inorganic particles.
The transition was sharp enough that it was not necessary to consider intermediate
layers between inorganics and rubber, which implies that TEM did not recognize the
interface layer of rubber (bound rubber or immobilized rubber layer) on the inorganic
surface.
The procedure for 3D-TEM measurements is outlined in Fig. 3.2 [8]. It is divided
into two steps, with a computer playing a major role in each step. Specimens of
approximately 200 nm thickness are prepared using a cryostat microtome. For the
fiducial marker method [14], colloidal gold particles of 10 nm diameter are spread
on the specimens before mounting it on a holder. While tilting the sample grid in
2° steps within the angle range from −70° to +70°, it is irradiated with the electron
beam to capture TEM images. The images are not simply 2D image slices, but 2D
projections of the mass–density distribution of the specimens. During the tilts, the
positions of the tilted images are aligned together with the axis of rotation being
calibrated by the fiducial marker and a computerized positioning of the relationship
between the images. The calibration due to the different thicknesses with the tilt
angles was automatically carried out during the measurement. The procedure up to
this point constitutes the first step.
The second step consists of computerized tomography, which is in principle the
same as a medical X-ray CT scan. The sequence of tilted images obtained is subject
to the Radon transform followed by the inverse Radon transform to reconstruct the
3D image in the real space (Radon transform is a kind of Fourier transform on