Читать книгу Diatom Microscopy - Группа авторов - Страница 17

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.