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].
1.6 Super-Resolution Optical Microscopy
Super-resolution optical microscopy is based on the photophysical and photochemical properties of specific fluorophores. Super-resolution optical microscopy can be divided into two main categories: 1) Methods where the nonlinear responses of fluorophores are used to shrink the point spread function in order to enhance image resolution. These methods include stimulated emission depletion (STED) microscopy [1.26], ground state depletion (GSD) microscopy [1.25], and reversible saturable optical fluorescence transitions (RESOLFT) microscopy [1.24]; 2) Methods where the stochastic emission of single fluorophores is used to register the location of each emitter at distinct times, thereby making it possible to distinguish two nearby fluorophores in time, These methods include photo-activated localization microscopy (PALM) [1.2], stochastic optical reconstruction microscopy (STORM) [1.58], and super-resolution optical fluctuation imaging (SOFI) [1.13].
Due to the nature of light diffraction, the image resolution of confocal microscopy is limited to roughly 250 nm, which makes it difficult to visualize silica-embedded proteins (ranging from ten to several hundred nanometers) via live-cell imaging. Super-resolution optical microscopy based on photoswitchable fluorescent proteins makes it possible to determine with a high degree of precision the location of proteins at the single-molecule level. Fusion proteins with silaffin-3 (tpSil3) are embedded in biosilica and permanently entrapped inside the valve region during biosilica formation [1.50]. PALM super-resolution optical imaging involves the expression of fusion proteins with photoswitchable fluorescent proteins, which can prevent the problem of silica limiting antibody access to the protein(s) of interest in STORM. Genetic transformation facilitates live-cell imaging of proteins associated with the cell wall, thereby making it possible to use the expression of GFP fusion proteins in Thalassiosira pseudonana to localize distinct regions of biosilica. Gröger et al. [1.22] used the inherently high labeling density of PALM to study proteins in diatom biosilica.
Prior to imaging, it is necessary to select suitable super-resolution probes for biosilica embedded fusion-proteins. Six fluorescent proteins (PATagRFP, PAmCherry1, PA-GFP, mEOS3.2, Dendra2, and Dronpa) have been activated efficiently in cytosol; however, only Dendra2, mEOS3.2, and Dronpa have been activated when embedded in biosilica. Fusing three fluorescent proteins to tpSil3 for PALM imaging has made it possible to achieve average localization precision of roughly 25 nm. Images are reconstructed (stacked) from more than 1000 frames to facilitate localization. Note that the production of pure biosilica requires the extraction of chloroplasts using a detergent-based buffer to eliminate their autofluorescence, which can hinder in vivo single-molecule localization microscopy. Figure 1.18a presents a PALM image of Dendra2 in the biosilica of the valve region of Thalassiosira pseudonana. Figure 1.18d shows the focal plane in the girdle band region. As shown in Figure 1.18b, the average full width at half maximum (FWHM) of 76.0 nm ± 4.6 nm (± S.D.) was derived from multiple line scans perpendicular to the biosilica cylinder (Figure 1.18a). The 76 nm measurement can be further deconvoluted to 53 nm ± 3 nm by taking
