Читать книгу Diatom Morphogenesis - Группа авторов - Страница 15

The beauty of diatoms was missed until the early, curious microscopists started observing ambiguous glassy microorganisms under their optical microscopes in the 18th century [1.22, 1.38]. Although the light microscope (LM) helped us to reveal the diatoms’ world, diatom frustules also helped the microscopists in developing and testing the quality and resolution of their optical microscopes [1.24, 1.25]. Since the nineteenth century, several works have been published on diatoms, its morphology, and taxonomy by remarkable workers including Kützing, Schmidt, Ehrenberg, Grunow, Hustedt, Krammer, Lange-Bertalot, and more (see references in Round et al. [1.38]). They described both living cells and clean frustules extensively using LM. The unique structure of diatom frustules under LM, with a variety of shapes and symmetries, has captured a wide interest; however, most of the diatom’s real art, at the nanoscale, was kept hidden. The limitations for observing frustule ultrastructure, especially details below 200 nm, were solved after the invention of the electron microscope [1.26]. In 1936, the transmission electron microscope (TEM) was used to capture the first micrograph of a diatom frustule [1.26, 1.27], using it as a test object for the quality and resolution of TEM. After that TEM was used to explore diatom ultrastructure. Following, the scanning electron microscope (SEM) was invented and used extensively as a more effective tool for exploring frustules morphology and ultrastructure [1.24, 1.28, 1.36, 1.38].

The details observed using the SEM and TEM reflected the beauty of diatoms when many hidden details became observable. For instance, some bright striae under an optical microscope appear as arrays of fine pores under the electron microscope (Figure 1.5a). It was, to some extent, a kind of revolution for diatom classification and taxonomy with the morphological details that became available down to 15 nm with SEM and below 10 nm with TEM (Figure 1.5b). Nowadays, the observation of diatom frustule morphology and ultrastructure using LM, SEM, and TEM became routine work for people working on ecology, environment, forensic, nanotechnological, and other applications that concern frustule ultrastructure, monitoring diatom species, and taxonomy.

Although 2D information can be collected from LM and TEM and the 3D-shape appeared under SEM, the information about the surface topology, internal ultrastructure, and siliceous element relationships within diatom frustules was missing. Therefore, more tools were evolved and involved in the exploration and understanding of the 3D complex ultrastructure of the frustule, which could be the reason for their various natural features, including unique photonic, mechanical, and hydrokinetic properties [1.9, 1.45]. The new tools include the atomic force microscope (AFM) and the focused ion beam SEM (FIBSEM) [1.32, 1.34, 1.35, 1.41].

Figure 1.4 Cleaned diatoms in valve (g, h–j, m–r, u, v) and girdle views (a–f, k, l, s, t, w, x). (a–e, g) Rhoicosphenia spp., frustules are clavate and strongly flexed, one valve is concave with long raphe branches and the other valve convex with shortened raphe, different depth pseudosepta visible; (f, k, l) Gomphonema spp. showing valve heterogeneity; (h) Gomphonella olivacea (Hornemann) Raben. (i) Planothidium lanceolatum (Bréb. Ex Kütz.) Lange-Bert, rapheless valve shown with asymmetrical central area containing depression; (j) Geissleria cascadensis (Sovereign) Stancheva and S. A. Spaulding, valves elliptic, with cuneate apices, coarse areolae, three pairs of annulae are present at each apex; (m) Planothidium delicatulum (Kütz.) Round and Bukht. Rapheless valve shown, lacking a central area and two middle striae spaced distantly. Cleaned diatoms in valve (g, h–j, m–r, u, v) and girdle views (a–f, k, l, s, t, w, x). (n) Gomphonema sp. valve heteropolar wider in the middle, axial area narrow, central area irregular outlined by two shortened striae and opposite to a single striae finishing with an isolated pore, striae parallel toward the headpole, radiate toward the foot pole; (o) Amphora ovalis, dorsal fascia visible and dorsal striae interrupted transapically by intercostal ribs; (p) Gomphonema micropus Reichardt lanceolate valve with headpole widely drawn out and wider than foot pole, striae radiate, central area unilaterally rectangular with shortened central stria, on the opposite side longer striae finishing with a stigmoid; (q) Navicula genovefae Fusey valve linear-lanceolate with rostrate broadly rounded apices, punctate striae radiate and curved, becoming nearly parallel at the apices, less dense around the well-defined central area; (r) Cocconeis placentula Ehrenb. Valves elliptic, striae radiate and interrupted by a hyaline ring positioned close to the valve margin, siliceous bridges (imbriae extending from valvocopula) visible; (s) Amphora pediculus (Kütz.) Grunow focus from dorsal site of two frustules; (t, u) Caloneis sp. on girdle view striae continue on valve mantle, on the linear valve view with rounded apices, axial area is narrow, broadening to a transverse fascia; (v) Navicula cryptocephala Kütz. Valve lanceolate with protracted apices and visible large, circular central area; (w) Mastogloia pseudosmithii Sylvia S. Lee, E. E. Gaiser, Van de Vijver, Edlund, and S. A. Spaulding, evenly sized partecta (chambers on the valvocopula) on both valves; (x) Navicula cf. tripunctata (O.F. Müll.) Bory. Scale bar, 10 μm. These micrographs were obtained and identified by KMM.

In 1992, the first observation of diatoms using an AFM has been done [1.33]. In general, AFM is used as an advanced tool to explore 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.