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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 the non-centric Pyrocystis fusiformis diatom. From [1.30] with permission of OSA.

On the other hand, the intense fluorescence of PDMPO bound to polymerizing silica under UV light illumination has provided new insights into the mechanisms underlying biological silicification. This method also makes it possible to study Si in natural diatom communities. Multiphoton microscopy can be used to reveal the fluorescence properties of Si-bound PDMPO as well as the 3D distribution of Si within diatom cells throughout the cell cycle. Figure 1.16 demonstrates one image section (2D image) of the respective labeled diatom, which can be visualized layer-by-layer and then reconstructed as a 3D image or z-stack animation. It is known that chlorophyll is commonly used as a contrast agent for imaging cell structures and evaluating cell toxicity. Two photon-excited fluorescent lifetime imaging (FLIM) can be used to assess the toxic effects of Thalasiosira weissflogii quickly and easily based on a statistical analysis of chlorophyll fluorescence decay (i.e., fluorescence lifetime change). By studying the fluorescence quenching of chlorophyll induced by the infrared two-photon excitation laser, the assessment of cadmium (Cd) toxicity based on the quenching effects can be well quantified and monitored. The characteristics of chlorophyll fluorescence in the time-domain could potentially be used as biomarkers to detect Cd toxicity levels without harming algae cells [1.75].

Figure 1.16 (a) Asterionellopsis glacialis and (b) Proboscia alata captured using a confocal multiphoton microscope (multiphoton process performed under a confocal microscope) at 2 y after staining with PDMPO for 24 h. These false color images were captured in black and white. For consistency with the epifluorescence microscopy images, Chl a appears in red and PDMPO appears in green. In the Asterionellopsis glacialis colony, the entire valve appears to be stained over the incubation period. This cylinder-shaped Proboscia alata diatom increases its length by adding girdle bands from the center of the cell. Newly deposited frustule parts are visible in green. From [1.35] with permission of John Wiley and Sons.

Figure 1.17 (a) Average fluorescence lifetime images of T. weissflogii exposed to different concentrations of MeHg; (b) Histogram analysis of fluorescence lifetime distributions; (c) Box analysis of fluorescence lifetime (reticular: mean value; cross: 1–99% of corresponding distribution; box size: 25–75% of the corresponding distribution; straight line: median) ((b) 0.0 nM at 0 h, before exposure; (a) 0.0 nM at 72 h, after exposure). Data are mean ± SD (n = 2). From [1.72] with permission of Elsevier.

The growth of diatoms is contingent on the environment. The presence of inorganic mercury (Hg (II)) and methylmercury (MeHg) has been shown to affect photosynthesis and population growth of diatoms. These effects can be observed using FLIM microscopy under two-photon excitation and flow cytometry (FCM). As shown in Figure 1.17, photosynthesis occurred in the presence of Hg (II) but in the absence of MeHg, as indicated by the extended fluorescence lifetime of the chlorophyll. Diatoms in the Hg (II) environment grew relatively slowly; however, the rate of division was unaffected. Diatoms in the MeHg environment presented slow growth as well as hindered cell division. Morphological changes in diatom cells exposed to Hg(II)/MeHg can be quantitatively measured from cell images acquired using multiphoton microscopy in conjunction with FCM data [1.72].

Diatoms have also proven highly effective in the production of petroleum substitutes and bioactive compounds. Researchers have developed an effective method referred to as intracellular spectral recompositioning (ISR) in which the absorption of blue light and intracellular emissions in the green spectral band enhance the utilization of light by the organisms. In Phaeodylum tricornutum diatoms, ISR can be chemogenically used with lipophilic fluorophores, or biogenically used to enhance the expression of green fluorescent protein (eGFP). In laboratory testing under simulated outdoor sunlight conditions, the biomass production of eGFP was 50% higher than that of the wild-type parental strain. Chlorophyll autofluorescence and eGFP fluorescence were detected via multiphoton excitation using a laser scanning microscope (FV1000, Olympus) [1.20].