Читать книгу Diatom Microscopy - Группа авторов - Страница 18

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-GFP^N and Sin1-GFP^C 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-GFP^N or Sin1-GFP^C. 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. The biosilica and organic matrix panels show bright field microscopy images (BF) and the corresponding epifluorescence microscopy images (EF) of material isolated from Sin1-GFP^N- or Sin1-GFP^C-expressing transformants. Scale bars in all images = 2 μm. From [1.32] with permission of Springer Nature.

Figure 1.12 Quantification of carbolines in healthy and oomycete-infected diatom cells. The spatial localization and accumulation of carbolines was observed with CLSM in healthy (upper row), early infected (middle), and late infected (lower row) cells. These microscopic images were captured in bright field (left) and the autofluorescence emissions of chlorophyll a (Chl a) (middle) and carbolines (right) were recorded under an excitation wavelength of 405 nm. Scale bars = 10 μm. From [1.68] with permission of Springer Nature.

Diatoms are non-toxic organisms. In fact, diatoms have been used as a nutritious food source and in the treatment of cancer. Functionalized diatomite nanoparticles (NPs) have also been used as non-toxic nanocarriers for the transport of small interfering ribonucleic acid (siRNA) for the treatment of human epidermal cancer (H1355) and colorectal cancer [1.54]. CLSM revealed the cytoplasmatic localization of vectors and gene silencing by delivered siRNA in cancer cells incubated with siRNA-conjugated NPs (see Figure 1.13). Diatomite NPs coated with B12 (cyanocobalami) have also been used as a tumor targeting agent. The functionalization of this material was examined using various analytical techniques and the synthesis of the organometallic luciferin analogue of cyanoco-balami was detected by CLSM. This B12 modified diatom was shown to facilitate the targeted delivery of water insoluble inorganic complexes to tumors [1.11]. Diatoms can also be used in molecular biology to research organisms with complex plastids, which are suitable fluorescent proteins for the in vivo analysis of protein localization. CLSM has been used to measure GFP fluorescence emission at wavelengths from 500 to 520 nm, while P. Tricornutum plastid autofluorescence is measured at wavelengths above 625 nm. The fluorescent protein mRuby3 has been developed as a tag for in vivo studies on the localization of proteins. More importantly, CLSM is ideally suited to co-localization experiments using mRuby3, in which mRuby3 fusion protein and GFP-tagged proteins are expressed simultaneously [1.37].

Figure 1.13 Evaluation of siRNA uptake and cellular internalization using confocal microscopy to estimate the ratio of red fluorescent cells (expressing Dy547) to total cells. The cells were imaged after being treated with siRNA*-modified diatomite nanovectors (first line) for 24 h with untreated cells as a control (second line). Cell nuclei and membranes were respectively stained with Hoechst 33342 and WGA-Alexa Fluor 488. siRNA was labeled with Dy547. Roughly 75% of the siRNA molecules in the first line are in H1355 cells, localized in cell cytoplasm rather than the nucleus. Scale bar = 20 μm. From [1.54] with permission of Elsevier.

The fact that folate receptors are highly expressed in the surface of many cancer cells means that folic acid can be used as a targeting ligand for the differentiation between normal and cancer cells. Rosenholm et al. [1.56] connected polyethylenimine (PEI) to diatom frustules. The attached amine group generated free electrons around the diatom frustule, which facilitates the connection to -N=C=S group, resulting in fluorescein isothiocyanate (FITC) fluorescence. The above was then connected to folic acid, FA, to form FITC/PEI/FA particles, which are visible under a fluorescence microscope. Because the internalization of the particle depends on the number of folate receptors, it was shown that 5-6 times more FITC/PEI/FA particles attach to HeLa cancer cells than normal cells (293), resulting in correspondingly strong green fluorescence, as shown in Figures 1.14a-1.14c. In addition, when mixing HeLa cancer with normal cells, the FITC/PEI/FA particles had a significant selective combination with cancer cells, showing these effects were particularly pronounced in cases HeLa cancer cells, as shown in Figure 1.14d.

Figure 1.14 Specific particle endocytosis of FITC/PEI/FA-functionalized silica nanoparticles. Normal cells (a) or HeLa cells (b) were left untreated or incubated with nanoparticles (10 g/ml) for 4 h, after which extracellular fluorescence was quenched using trypan blue. The endocytosed particles with FITC label (green) inside CMAC-labeled (blue) cells were detected via confocal microscopy (a and b) or flow cytometry (c). Mean fluorescence intensity (MFI) values of FITC were normalized to particle endocytosis in HeLa cells. (d) Specific endocytosis of FITC/PEI/FA-functionalized silica nanoparticles in co-culture of HeLa cells (labeled using blue CMAC) and 293 normal cells (labeled using CellTracker Red). Scale bar = 30 μm. The results are representative of two independent experiments detected using a confocal microscope. From [1.56] with permission of American Chemical Society.