in this chapter. First, we discuss light microscopy in Section 1.2, which is not associated with fluorescence. It forms the image of diatoms using basic light characteristics such as scattering, diffraction and interference. Then, we discuss fluorescence microscopy and confocal microscopy in Section 1.3 and Section 1.4 respectively, which can provide images with high contrast, specificity and selectivity using fluorescent probes or the probes synthetized with diatoms. Next, we discuss multiphoton microscopy in Section 1.5, which enables high-contrast, label-free and deep imaging, as a patch up to the weakness of fluorescence-based microscopy. At last, we discuss the recent advanced imaging technique, super-resolution optical microscopy, in Section 1.6 for the study of diatoms.
1.2 Light Microscopy
1.2.1 Phase Contrast Microscopy
The sensitivity of diatoms to variations in aqueous growth conditions (e.g., temperature, salinity, oxygen content, and pH) makes them excellent indicators of changes in water ecosystems (e.g., eutrophication, pollution and acidification) [1.15, 1.53]. Figure 1.1 taken from phase contrast microscopy illustrates the diversity of diatoms in Baryczka stream (Poland), which were used as an indicator of water quality [1.44]. Besides, phase contrast microscopy has been used to study Azpeitia africana (a typical warm water diatom species) as an indicator of water temperature in the Pacific Ocean [1.71]. Diatoms are microscopic algae enclosed between two valves of hydrated amorphous silica. As shown in Figure 1.2, these intricate structures present a symmetric pattern of pores at the micrometer- and nanometer-scale. The highly symmetrical 3D geometry of the silica skeletons produces peculiar optical characteristics, similar to those produced by photonic crystals [1.14, 1.21]. This similarity has provided an interesting point of view in optical properties for light guiding and optical transducing in the electromagnetic field [1.8].
Quantitative phase imaging (QPI), as an extended form of phase contrast microscopy, makes it possible to measure the complex transmission function (i.e., the discrete Fourier transform (DFT) of sample under Rheinberg illumination, which contains amplitude and phase information) of diatom samples. Color contrast can be applied to images by introducing optical staining via an amplitude filter placed in the illumination path. Figure 1.3 presents quantitative phase images of diatoms obtained via digital holographic microscopy [1.17]. It was obtained by Rheinberg filter simulation on the back focal plane of a microscope condenser lens positioned for Kohler illumination that occurs in the discrete Fourier transform (DFT) domain. Note that the nine point sources at the focal plane of the condenser, as shown in Figure 1.3c, are transformed to plane-wave illumination on the sample. Then, the resulting image is formed by the sum of the nine independent image intensities, of which each image corresponds to the spatial filtering to different regions of the DFT of the complex transmittance function, as shown in Figure 1.3e, which is done prior to an inverse DFT for returning it back to the spatial domain as an image. Furthermore, a similar approach was reported that a contrast was produced in image intensity, which is correlated with the phase delay introduced by the specimen. This method allows for a real-time imaging of cellular morphology with nanometric axial resolution. Figure 1.4 shows two diatom cells recorded by QPI, in which the color filters, as a type of optical staining that controls light amplitude at specific wavelengths, placed in the illumination path are used to provide color contrast to images of phase objects. They are color-coded according to small values of local spatial frequency/ray angle, which make it possible to visualize the details of diatoms [1.18]. These results clearly demonstrate the applicability of QPI for the label-free color staining of biological samples.
Figure 1.1 Selected diatoms taxa in the Baryczka stream obtained using phase contrast microscopy. Note that these images were used as an indicator of water quality. From [1.44] with permission of Bentus.
Figure 1.2 Comparison of diatom defects and man-made photonic crystal fiber. The circular holes and hexagonal lattice of the diatom is similar to the structure of a hexagonal lattice solid-core photonic bandgap fiber used to confine light via band gap effects. From [1.8] with permission of Elsevier.
1.2.2 Differential Interference Contrast (DIC) Microscopy
In differential interference contrast (DIC) microscopy, two cross-polarized light beams pass through a specimen and then combine to form interference fringes indicated by the thickness and birefringence of features of interest. DIC microscopy has been used in forensic medicine to characterize the morphology of diatoms, reproductive biology to characterize sperm cells and hematology for the early discovery of cancer cells [1.46]. This method provides high-resolution images with good contrast without halos around structures, even when imaging thick samples, in contrast to phase contrast microscopy. However, tiny changes or fine structures, which are below the resolution limit, might be invisible when imaged by conventional photography. Until 1980s, electronic contrast enhancement capabilities of video cameras were found to visualize 25-nm diameter micro-tubules by DIC microscopy with the resolution nearly an order of magnitude smaller than the diffraction limit [1.1, 1.59]. In the natural environment, phytoplankton and bacteria live in close proximity. Highresolution video-enhanced differential interference contrast (VEDIC) microscopy has been used to characterize the relationship between marine diatoms and the bacterium L. monocytogenes. As shown in Figure 1.5, the relationship between listeria and the benthic diatom Navicula sp. is parasitic in nature, whereas the relationship between the listeria and the planktonic diatom Phaeodactylum tricornutum is protocooperative (protocooperative: the co-cultivation of planktonic diatom with any of the L. monocytogenes strains can enhance the growth of both the diatom and the listeria cells.) [1.65].
Figure 1.3 Quantitative phase image of diatom cell recorded using 20 x/0.45 microscope objective. Shown here are the (a) intensity and (b) phase images of the complex transmittance. (c) Illustration of simulated Rheinberg filter located at the rear focal plane of a microscope condenser lens. The filter comprises eight red point sources located around a center green point source. (d) Resulting image from simulated Rheinberg illumination. (e) Filtering implemented in the DFT domain, for each of the independent point sources in the filter. From [1.17] with permission of OSA.
