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      Investigation of Diatoms with Optical Microscopy

       Shih-Ting Lin1, Ming-Xin Lee2 and Guan-Yu Zhuo1,2*

       1Integrative Stem Cell Center, China Medical University Hospital, Taichung, Taiwan

       2Institute of New Drug Development, China Medical University, Taichung, Taiwan

       Abstract

      Diatoms are eukaryotic microalgae occurring in the water column as phytoplankton and on the ocean bed as benthic microalgae. Diatoms are similar to plants in the fact that they use photosynthesis, but differ in terms of evolutionary classification. More than 10,000 diatom species have been formally described, based primarily on the unique patterning of their silica shells. Over the last two decades, diatoms have attracted considerable attention for their potential use in synthesizing bio-functional materials due to the advantages of low production cost, prevention of using toxic chemicals and producing hazardous waste and intricate 3D hierarchical structures applicable for photonic applications, biosensors, catalysis, adsorption, and drug delivery systems. All such applications require a comprehensive understanding of the diatom structure, from the microscale to the nanoscale. Optical imaging provides spatial resolution at the sub-micrometer scale without harming the specimens. Image post-processing and reconstruction also make it possible to render the structure of samples in 3D via optical sectioning. In this chapter, we explore the various facets of optical microscopy within the context of diatom research and the applicability of this work to eco-environmental science and biomedicine. In the following sections, we respectively address light microscopy, fluorescence microscopy, confocal laser scanning microscopy, multiphoton microscopy, and super-resolution optical microscopy.

      Keywords: Light microscopy, phase contrast microscopy, differential inteference contrast microscopy, darkfield microscopy, fluorescence microscopy, confocal microscopy, multiphoton microscopy, super-resolution optical microscopy

1.1 Introduction

      Diatoms present a three-dimensional (3D) hierarchical structure featuring a nano-patterned porous silica shell (i.e., frustule), which is surprisingly similar to artificial photonic crystals in its ability to manipulate light [1.21, 1.67, 1.73, 1.77]. Diatom frustules comprise two parts, an upper cover (epitheca) and a lower cover (hypotheca), both of which possess a shell surface (valve) and several silicon segments referred to as a girdle band. The upper and lower covers overlap around the shell ring in a manner similar to a soap dish protecting the cell content within. There has been considerable research into the synthesis of porous silica nanoparticles (NP_S), due to their large surface area and biocompatibility, which makes them ideally suited as sensors, optical devices and drug delivery systems [1.57]. The diatom frustule is a nano-patterned cell encasement made of amorphous biosilica, which occurs in an astonishing range of structures. This has prompted research into the fabrication of functional materials using diatom frustules cultivated under artificial conditions. The formation of silica shells involves highly phosphorylated proteins, long-chain polyamines and carbohydrates [1.33] in a series of biomineralization processes, many of which can be replicated in test tubes. The accumulation of diatoms on the ocean floor results in the formation of a sedimentary mineral referred to as diatomite, a highly porous silicate with low density, high absorption capacity and high surface roughness. Researchers are developing methods by which to modify the surface properties of diatomite particles with specific functional groups or combine diatomite with fluorescent particles for use in microscopic imaging applications [1.45, 1.69].

      A number of microscopy techniques are used in the study of diatoms. Electron microscopy provides outstanding resolution, whereas optical microscopy permits the real-time dynamic observation required for biological research [1.23, 1.31]. In the last century, the historical development of optical microscopes has focused primarily on improving image contrast, leading to the development of phase contrast microscopy, differential interference contrast (DIC) microscopy, darkfield microscopy, and polarized light microscopy. Since the advent of dye-labeling technology [1.34], fluorescence microscopy has become the most common imaging tool in biological research; however, fluorescence microscopy is prone to interference from out-of-focal plane scattering, which prevents 3D imaging. Fluorescent molecules are also prone to photobleaching under irradiation by strong light [1.51].

Confocal laser scanning microscopy (CLSM) involves the focusing of a low-energy laser source on a small spot (to prevent photo-bleaching) and the placement of a pinhole in front of the photodetector (to block out-of-focal plane signals) [1.40, 1.48]. In addition to enhancing the contrast and resolution of images, this approach enables the observation of samples at specific depths layer-by-layer (i.e., optical sectioning) as well as the reconstruction of images (i.e., layer stacking) to render the sample in 3D. CLSM can be used to observe cell samples of <50 μm; however, it is inapplicable to samples of >100 μm, due to limited light penetration resulting from the absorption and scattering of light at visible wavelengths. Note that applying light of higher intensity to compensate for a loss of photons by the confocal pinhole and imaging biological samples for a long time with visible wavelengths can greatly exacerbate phototoxicity and/or photobleaching.

      Since the advancement of ultrafast laser technology, the shortcomings of CLSM have largely been resolved by equipping laser scanning microscopes with ultrafast lasers. This method (referred to as multiphoton microscopy) [1.3, 1.12, 1.38] typically uses near-infrared (NIR) excitation to minimize the water absorption in the tissue and thereby increases penetration depth to hundreds of micrometers. Importantly, the multiphoton effect only occurs at the focal plane (i.e., inherent optical sectioning) without the need for an additional pinhole to filter the out-of-focal plane scattering and the problem of emitted photon loss shown in CLSM. In addition, the use of a laser source with high peak power and low average intensity allows imaging over extended durations without inducing phototoxicity or photobleaching. Based on these advantages, multiphoton microscopy can be used for the 3D imaging of thick biological tissues in vivo or ex vivo. In the last decade, super-resolution optical microscopy [1.2, 1.26, 1.41] honored by the Nobel Prize in Chemistry 2014 is devised to overcome the diffraction barrier of light [1.28, 1.47], providing a greatly improved resolution on several tens of nanometer scale (i.e., an order of magnitude improvement over conventional optical microscopy). In addition, it is demonstrated in the far-field at arbitrary biological states and no need to pretreat the sample prior to investigation, which are the advantages over electron microscopy. Associated techniques mainly rely on fluorescence microscopy and advanced fluorescent probe [1.19, 1.66] with a fast on/off switching rate and better stability. It promotes the progression of single-molecule detection and cell and molecular biology in visualizing the fine structures and dynamic processes of single molecules inside cells or tissues.

In the following sections, we provide a comprehensive understanding of the application of the above-mentioned microscopy methods to the study of diatoms 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 Скачать книгу