microscopic image showing D24 systems after incubation with fluorescent 50 nm yellow microspheres. Subsequent fluorescence analysis with background-free images revealed device localization, selectivity, and specificity. From [1.45] with permission of Springer Nature.
1.4 Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy (CLSM) has been used with hyperspectral analysis to characterize the optical properties of diatom frustules [1.55]. Enhanced light transmission through the diatom frustule was observed at roughly 636 and 663 nm, which respectively match the maximum light absorption of chlorophyll a and c, as shown in Figure 1.9. This is a clear demonstration that the efficient light trapping of the frustule can be attributed to strong asymmetry between cribrum and foramen pseudoperiodic structures, which prevents the backscattering of transmitted light resulting in a corresponding increase in light absorption.
In terms of diatom ecology, Buhmann et al. [1.4] established a system for the incubation of pure phototrophic biofilms in the laboratory, with the aim of elucidating microbial population dynamics and community interactions associated with biofilm development. That system proved effective in the growth and monitoring of three strains of Planothidium sp. strain 8c isolated from epilithic (epilithic: growing on rock surfaces) biofilms of an oligotrophic freshwater lake for co-cultivation with axenic (axenic: the state of a culture where only a single species is presented that is free from other cultivatable organisms) diatom phototrophic biofilms. Automated photometric monitoring of biofilm density under controlled illumination revealed a 3x increase in biofilm turbidity (turbidity: degree of light attenuation) when co-cultured with Planothidium sp. strain 8c, indicating the co-existence the chlorophyll autofluorescence and stained-bacteria. This type of system can be used to evaluate diatom biofilms and study the interactions in diatom-bacteria biofilms. Diatom diazotroph (diazotroph: mostly bacteria and archaea that can transform nitrogen gas into a usable form such as ammonia) association (DDA) is among the oldest planktons, which lives on the surface of the sea at elevated temperatures under high CO2 levels, such as those predicted by most models of global warming. DDAs are marine plankton that combine with bacteria with fixed nitrogen (N2) from a variety of diatom genera. The development and distribution of DDA can be observed using molecular genetics methods to assess cells collected in the field via single-cell CLSM. The distribution and evolution of DDAs and their phylogenetic diversity can be characterized using confocal analysis with 3-D imaging re-evaluation, as shown in Figures 1.10a-1.10c [1.5].
Figure 1.9 Hyperspectral analysis of a single valve of Coscinodiscus centralis oriented with the foramen side upwards and the direction of the incident light indicated by the white arrow (a). The wavelength regions (green and red) where transmission spectra (b, c) differing from the light source are detected. Close view of hyper-map in a (d). Close-up view of composite image in a (e). The yellow lines in (d, e) indicate the transition between the foramen and cribrum layers of the valve, with the honeycombed structure in the middle. From [1.55] with permission of Springer Nature.
McNair et al. [1.39] proposed a method for quantifying biogenic silica (bSiO2) production, in which the fluorescent dye PDMPO is used to dissolve diatom frustules. PDMPO is incorporated in SiO2 deposition vesicles (SDV) and newly formed diatom frustules. The use of PDMPO to measure bSiO2 production by a single diatom cells requires quantification of single-cell fluorescence and accurate calibration of the relationship between the incorporated PDMPO and the amount of newly polymerized silica. CLSM is used to detect single diatom cells. Improvements in PDMPO labeling have made it possible to progress from qualitative assessment to quantitative measurement of bSiO2 in newly deposited diatom communities and single diatom cells. On the other hand, diatoms are commonly divided into two breeding methods of which the silicification process start from the SDV inside the cell membrane. However, there is few discussions on the SDV membrane protein of diatoms or other silica-forming organisms. For this, Kotzsch et al. [1.32] discovered a unique SiMat7-like protein, as the organic component of diatom silica, which is significantly different from other proteins in the secondary structure. It was also proved that SiMat7 is the original member of a new type of silica biomineralization protein family, named as Siliconnin-1 (Sin1). After protein analysis, Sin1 sequence was divided the into two regions, an NQ (asparagine and glutamine)-rich domain and a cytosolic domain. By connecting GFP to different positions, it formed into Sin1-GFPN and Sin1-GFPC shown in different parts of living cells, which can be observed under CLSM (see Figure 1.11). The NQ-rich domain is flipped out of the membrane, and the cytosolic domain remained between the cells, manifesting that Sin1 is a transmembrane protein.
Figure 1.10 Laser confocal microscope images of H. hauckii-R. intracellularis symbiosis simultaneously excited by laser light at frequencies of 488 and 561: (a) z-stack image, (b) orthogonal views (xy, xz, yz) and (c) processed using the Contour Surface tool in IMARIS v.8.1 (Bitplane). White arrows indicate the fluorescence of the chloroplasts in xz and yz. Chloroplasts appear in green, and cyanobacteria trichome (filament) in orange. Scale bar = 5 μm. From [1.5] with permission of Oxford University Press.
Antifouling coatings can be fabricated by natural products that are dissolved in biofilms of bacteria and diatoms. A new method combining a high-throughput microplate reader, CLSM and nucleic acid staining has been developed to assess the activity of such coatings in terms of marine biofilm formation [1.60]. One approach to solving the problem of marine plastic marine debris (PMD) involves the use of microbes and biofilms. The combinatorial labelling and spectral imaging–fluorescence in situ hybridization (CLASI-FISH) tool makes it possible to visualize specific groups of microbes and elucidate their interactions with substrate surfaces, including microbial and diatom biofilms, which serve as polymer hydrolyzers capable of degrading PMD [1.6, 1.42, 1.62, 1.76]. Note that the marine ecological environment features numerous parasitic relationships. Vallet et al. [1.68] demonstrated that the flagellate marine oomycete (oomycete: commonly known as water mold, a kind of eukaryotic microorganism that is very similar to fungi. It is not organized with chlorophyll and thus does not perform photosynthesis. It requires nutrients after in vitro decomposition and then absorbs again) L. coscinodisci can infect Coscinodiscus granii diatoms. Figure 1.12 presents the infection mechanism in which algal carbolines accumulate in the reproductive form of the parasite, as revealed by a single-cell analysis based on AP-MALDI-MS and CLSM.
Figure 1.11 Live cells, biosilica and biosilica-associated organic matrix from transformant strains expressing Sin1-GFPN or Sin1-GFPC. The fusion proteins were expressed under control of the endogenous Sin1 promoter and terminator sequences. The ‘Live cell’ panels show confocal fluorescence images (z-projection) of individual cells in girdle view (left panel and third panel from the left) and in valve view (second panel from the left). Green color indicates the GFP fusion proteins and the red color is caused by chlorophyll autofluorescence.
