biosilica and organic matrix panels show bright field microscopy images (BF) and the corresponding epifluorescence microscopy images (EF) of material isolated from Sin1-GFPN- or Sin1-GFPC-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.
1.5 Multiphoton Microscopy
Multiphoton (MP) imaging is used to create 3D images based on nonlinear optical effects, such as harmonic generation (HG) and multiphoton fluorescence (MPF). In the following case, two third-order nonlinear optical processes, three-photon excited fluorescence (3PEF) [1.27, 1.63, 1.70] and third-harmonic generation (THG) [1.7, 1.64, 1.74], were combined to investigate diatom structures. The former mechanism is using longer wavelength (typically longer than 1300 nm) to excite fluorophores when three incident photons with the same energy are absorbed simultaneously. Then a new photon with the energy slight smaller than the sum of the three photons is emitted from the fluorophores. Conversely, in the latter mechanism, a new photon with the energy equivalent to the sum of the three photons is emitted from the surface/interface at with a refractive index mismatch is shown due to Gouy phase shift. Thus, THG is used to image the morphology of sample. The main advantage of three-photon microscopy is the much longer penetration depth (typically longer than 1mm) and intrinsic optical sectioning capability, which facilitates to image thick biological tissues and form a high-quality 3D image. In Figure 1.15, an Er3+-doped femtosecond fiber laser (wavelength of 1560 nm) has been used in conjunction with carbon nanotubes (CNTs) to observe chlorophyll fluorescence in plant leaves and diatoms based on the 3PEF. And the lipid cell layers inside the chloroplasts were visualized by THG. The penetration depth can be increased by setting a high pulse energy and short pulse duration. In the experiment, increasing the wavelength to 1650 nm further enhances imaging depth and reduces the likelihood of sample heating due to very low absorption of light at that frequency by biomolecules. Furthermore, the absorption rate of water at about 1650 nm is several times smaller than the 1560 nm of the incident light [1.30].
Figure 1.15 Left: Multi-photon image of living centric diatoms Coscinodiscus wailesii. As with the fresh leaf of mesquite, a desert plant originated in Arizona, we observed strong THG signal (green) from lipid cell layers inside the chloroplasts and 3PEF signal (red) from chlorophyll. Right: 3D view of a 3PEF and THG image of a living non-centric diatom Pyrocystis fusiformis. Inset: Optical image of
