Diatom Microscopy. Группа авторов

Figure 1.5 Images illustrating the relationship between the bacterium L. monocytogenes strain P and the benthic diatom Navicula sp.: (a) first stage, (b) second stage, (c) third stage, and (d) control. The scale bars = 10 μm. From [1.65] with permission of Springer Nature.

      1.2.3 Darkfield Microscopy

The widespread use of silver nanoparticles (Ag NPs) in industrial applications has prompted concerns about their effects on the environment [1.16]. Researchers have used dark field images to investigate the uptake of Ag NPs by the marine diatoms Cylindrotheca fusiformis and Cylindrotheca closterium and the biological effects on the organisms. As shown in Figure 1.6, Ag NPs detected throughout the diatom cells caused dramatic morphological changes within the cells but had little effect on the cell walls [1.49].

Figure 1.6 Dark field micrographs of control cells and cells exposed to Ag NPs. Ag NPs appear as bright, luminescent spots due to surface plasmon resonance. Scale bar = 10 μm. From [1.49] with permission of John Wiley and Sons.

Figure 1.7 (a) Dark field image of single valve of C. wailesii diatom and (b) corresponding fluorescence macroscopic image. From [1.9] with permission of Hindawi.

At the macroscale, the wide bandgap of amorphous silica does not produce photoluminescence; however, dark field and photoluminescence imaging have shown that nanoscale silica particles exhibit photoluminescence at appropriate excitation wavelengths [1.52]. In addition to photoluminescence from nanostructured silica and diatom frustules, there is also a contribution of organic residuals incorporated in the porous silica matrix. Figure 1.7 presents dark field and green fluorescence images at excitation wavelengths of 450-490 nm [1.9]. It is noted that the photoluminescence process converts the DNA-harmful UV radiation in blue radiation. Due to the maximal efficiency of photosynthesis is in the red spectral region, the green fluorescence appeared in the macroscopy images. These results indicate that there are three mechanisms by which the frustule protects the living diatoms from UV radiation: light absorption, light-collection inhibition and wavelength conversion.

1.3 Fluorescence Microscopy

Living diatoms present fluorescence from the frustule (frustule photoluminescence), chloroplasts, and lipid layers (autofluorescence). Both forms can be observed under a fluorescence microscope under an excitation wavelength of 450-490 nm, as exemplified by the C. wailesii diatom in Figure 1.7 [1.9]. The enormous number of diatom species distributed throughout the world makes them an ideal vehicle by which to monitor water quality [1.29] and changes in streams and rivers over extended timespans [1.39]. In the last decade, researchers began using transgenic technology to alter diatoms with the aim of extending this analysis. For example [1.36], in a poor growth environment, diatoms will produce alkaline phosphatase, of which the promoter of alkaline phosphatase of P. tricornutum is very active. It can be used to connect the green fluorescent enzyme behind the promoters, so that in the absence of phosphate, the red autofluorescence from chloroplasts in diatoms illuminated by blue light would be changed into green autofluorescence. On the other hand, the fluorescent dye, PDMPO (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy) phenyl)oxazole) combines with the silicic acid used by diatoms in the formation of silicified cell walls and the incorporation ratio of PDMPO and biosilica remain nearly constant for an extended duration to 2 years [1.35, 1.39]. The images in Figure 1.8 were taken between 1 and 2 years after the sample was mounted on glass slides. They clearly demonstrate the persistence of PDMPO fluorescence in illustrating the fine structure of valve areolae and striae using a fluorescence microscope under UV light excitation. With this method, the silicification of an entire diatom community can be quantified by converting PDMPO incorporation to silica production using diatom bSiO2:PDMPO incorporation ratios [1.39].

The rate of diatom silicification depends on environmental factors, such as water temperature, pH and depth. Znachor and Nedoma [1.78] used fluorescence microscopy for imaging and measuring single-cell PDMPO fluorescence to observe relative differences in silicification among diatom species. The acquired images showed that the fluorescence signals from diatoms and chloroplasts are easily discerned, which can be used to confirm an entire community in a silicified state. Onesto et al. [1.45] cultivated diatom frustules with Au NPs, which is abbreviation as D24 metallic NPs, at a ratio of 1:10 ratio for 10 minutes in order to use the diatoms to detect chemical pollutants (mineral oil). Under a fluorescence microscope, the diatoms absorbed light at a wavelength of 441 nm and emitted a fluorescence signal at 485 nm, as shown in Figure 1.8c. This system proved highly selective, specific and sensitive. Bovine serum albumin (BSA) can be detected in simple diatom frustules, and Au nanoparticles in D24 systems highlight the presence of the aromatic components of BSA at 1392 cm^-1 and in the 1556–1576 cm^-1 band, which are confirmed by surface-enhanced Raman spectroscopy. This method can be used to detect BSA at low concentrations, for use in bioengineering, medicine and pollution monitoring. Furthermore, Delalat et al. used genetic engineering to promote the expression of the IgG-binding domain of G protein on the surface of diatom frustules (Thalassiosira pseudonana) that was treated as drug carriers for the selective killing of neuroblastoma and B lymphoma cells. Thus, diatoms can be used as a target for drug delivery and absorption in the body, where the therapeutic effects can be examined and monitored under a fluorescence microscope [1.10]. The polyunsaturated aldehydes (PUAs) secreted by diatoms also have anti-cancer effects. Clementina et al. [1.61] demonstrated that PUA substances killed lung cancer cells and colorectal cancer cells without adversely affecting normal cells.

Figure 1.8 Images of new silicon deposits and chlorophyll a (Chl a) of coastal and subarctic diatoms under fluorescence microscopy. (a) PDMPO-stained diatom valves in the blue emission spectrum images using a DAPI filter. (b) Newly deposited valves exhibited an intense yellow-green fluorescence, and Chl a may in some cases be visible simultaneously in red. From [1.35] with permission of John Wiley and Sons. (c) Fluorescence 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