diatom ultrastructure providing information in the Z-direction, with the ability to understand the surface topology of the frustule parts with a nanoresolution. For instance, AFM observations of Coscinodiscus sp. clean valves revealed a distinct dome topology for the cribellum, which was not observed before [1.34]. At the beginning of the current century, AFM was used in several works for understanding the nanoscale ultrastructure and topology of frustule surfaces in a 3D manner. Today, AFM is also used to explore the organic envelope, micromechanical properties, and to understand the biomineralization processes of diatom frustules [1.35]. Luis et al. [1.35] can be considered a good review for starting AFM studies on diatom frustules.
Figure 1.5 (a) SEM of a single cleaned partially open frustule, two overlapping valves, of Nitzschia palea(Kützing) W. Smith, and scale bar is 5 μm. The rows of pores (striae) that observed here cannot be observed under LM for this species. (b) TEM of a close-up in Navicula sp. valve showing the hymenate pore occlusions that will not be observed under SEM; scale bar is 200 nm. These micrographs were obtained and identified by MG.
Figure 1.6 A cross-section at the center of Coscinodiscus sp. cell collected and treated while binary fission process was in progress, fabricated and captured by FIB-SEM. Reproduced from Xing et al. [1.42] under a Creative Commons Attribution 4.0 International license.
Furthermore, diatom valves seem to have a complex inner ultrastructure that cannot be understood completely by observing the internal and external view of a given valve surface using the previously mentioned tools. Although the multilayer, multiscalar porosity can be observed easily using such techniques, the internal anatomy and relations of the siliceous elements of the frustule cannot be understood [1.41]. It was usual to wish that the observation of a broken valve or girdle band at the right site and right angle would help, otherwise, the complex inner structure remained unseen [1.41].
Thus, another advanced method was required for understanding the inner structures and spatial relationships of the siliceous elements of a given diatom frustule. The FIB-SEM was introduced as a solution for such a problem by cutting the diatom frustule parts at nanoresolution to reveal the inner complex ultrastructure of a given valve or frustule (Figure 1.6) [1.41]. Suzuki et al. [1.40] was the first work introduced using FIB-SEM for making a cross-section in diatoms. Only a few articles are available using FIB-SEM and the field is still growing. The acquired data using FIB-SEM could be used to reconstruct the overall 3D geometry of diatoms to carry out further computational simulations necessary for diatom nanotechnology applications.
Table 1.1 A summary of the major tools used to study diatom frustule morphology and its ultrastructure.
| LM | TEM | SEM | AFM | FIB-SEM | |
| The date of first known observation of diatoms using the tool | Anonymous, 1703 [1.22] | Krause, 1936 [1.27] | Mid of 1960s [1.24] | Linder et al., 1992 [1.33] | Suzuki et al., 2001 [1.40] |
| Up-to-date resolution | The maximum resolution of the common compound optical microscope can be around 200 nm. Recently, the resolution was enhanced (down to 97 nm) using special kind of lenses [1.37]. | Up-to-date, the highest TEM resolution could be down to 50 picometer or even lower [1.29]. | The details less than 15 nm was not resolved under most of SEMs. Recently, an outbreak has been achieved, and the resolution of SEM could be below 1 nm [1.39]. | Recently, the resolution can be below 1 nm. | Having SEM as the microscope part of the device. Thus, the resolution is dependent on this SEM. |
| When we should use? | Observation of the presence or absence of diatoms in a sample. Identification of diatoms on the genus level. Enumeration of diatom frustules for different purposes. | Observation of the fine porosity (mesopores) present in some genera, like raphid pennates (Figure 1.5b).Observation of thin cross-sections in a valve or a girdle band.Observation of the cytoplasmic components of thin cross-sections of living cells (living cells anatomy). | Observation of the outer ultrastructure including most porosity.Observation of the overall 3D ultrastructure of the frustule or different parts.Identification at the species and subspecies level. | Observation of the 3D topology of a diatom frustule or its components.Measuring forces related with both living diatoms and its cleaned frustules. | Understanding the inner ultrastructure of diatom frustule or its parts by cutting cross-sections through it.Observation of the siliceous elements structural relations within the frustule.Observation of the whole 3D ultrastructure of the frustule via the 3D reconstruction. |
| The disadvantages | The observations for most of the ultrastructure details will be limited. Either the girdle view or the valve view will be available. | Only the tiniest parts of the valve, like pore occlusions, will be observed.The high energy electron beam may damage some sensitive samples, so it should be used wisely. | The samples must be coated with a conductive layer, which in turn could change the nano texture of the frustule silica and probably pore sizes, thus the thickness and smoothness of the conductive layer should be optimized and be thin as possible without getting nanoparticles on the top.The high energy electron beam may also damage some sensitive samplesThe regular resolution keep the pore occlusions of very fine porosity (below 10 nm) hidden. | The frustules must fix to the substrate before measuring.A very sensitive tool with complicated precautions to follow to get the desired results. | This technique sometimes needs more sophisticated preparation of the samples and more sophisticated work to reconstruct the frustule or its parts, however it worth.Related with the presence of the device, which usually is not available for all research groups. |
Finally, all the techniques mentioned were summarized in Table 1.1 to help beginners and students choose between different tools on-demand.
1.3 Diatom Frustule 3D Reconstruction
Toward the complete understanding of the 3D structure of a given diatom frustule, a comprehensive 3D model can be created from the data collected from different characterization techniques. This approach, which is designated as the 3D reconstruction of diatom frustules, can be used for different purposes but is mainly for computer modeling. Oncoming tools for the 3D reconstruction of diatom frustules are FIB-SEM [1.32, 1.42] and digital holographic microscopy (DHM) combined with SEM [1.30]. The combination of DHM and SEM or AFM might give the ability to