Diatom Morphogenesis

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.

Finally, all the techniques mentioned were summarized in Table 1.1to help beginners and students choose between different tools on-demand.

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 model and visualize microscopic 3D objects with a high resolution in all directions [1.30]. Hildebrand et al . [1.32] introduced the ability for the 3D reconstruction of subcellular architecture using FIB-SEM with new insights into the architecture and synthesis process of both the siliceous and organic components inside the cell. Xing et al . [1.42] is an inspiring reference for the 3D reconstruction of diatom frustule using the 2D image series resulting from FIB-SEM.

1. Fresh samples, if available, which means the sample is not diatomite (i.e., the fossil form of diatom frustules) [1.31], allow evaluation of the physiological state of the population at the time of collection. The features of living cells might help in the identification process, through the descriptions of colonial forms, cell attachment, extruded materials, plastids, and nucleus position (for instance see Figures 1.1 and 1.2). Since diatom frustules are rigid silica, they appear from two different viewpoints, the girdle view (from the side) and the valve view (from the front) under LM Figure 1.4. Some genera are easily identifiable in girdle view, such as Mastogloia and Amphora pediculus (Kütz.) Grunow, others are not. Lately, confocal microscopy uses are very promising [1.3, 1.13], also combining microscopic and molecular information allowed reclassification of a population in culture annotated as a radial centric species related to Leptocylindrus danicus Cleve, as an araphid pennate species in the staurosiroid lineage, within the genus Plagiostriata [1.14].

2. The selection of the suitable cleaning procedure, depending on the source of diatoms, is a crucial step to extract the siliceous frustule parts. Routinely, diatoms are identified and enumerated without their protoplasts where striae morphology & number, raphe shape, and other morphological characteristics could be verified for identification (details in Figures 1.3b and 1.4). Among the best guides for the cleaning process could be Wang et al . [1.43]. More gentle cleaning methods (e.g., H 2O 2method) should be considered in case we need to preserve mesopores (i.e., fine pores with diameters ranging from 50 to 3 nm that present on pore occlusions, which cover the areolae from inside or outside [1.48]) especially within genera, such as raphid pennate diatoms (Figure 1.5b).