Scanning Near-Field Optical Microscopy of Living Cells in Liquid, Elaboration of New SNOM Probes and Detection Methods

The aim of the present PhD thesis is the elaboration of new, home-made Scanning Near-field Optical Microscope (SNOM or NSOM), and the demonstration of its great potential for high-resolution topography and fluorescence investigations of soft and solid samples, as well as the possibility to work in air and liquid environments showing high-resolution topographical and fluorescence abilities to investigate soft and solid samples. The first chapter starts from with the description of the general idea of SNOM and with a brief introduction into to the history and theory of optical image formation, and leading to the resolution limits in conventional optical microscopy. The following section will discuss about the different state-of-the-art different SNOM configurations and theirs peculiarities. The second chapter discusses the SNOM instrumentation and its operation in liquid. The central idea of the proposed approach is the application of the "joint recipient principle" method. Then we will describe the double-resonance principle of the SNOM sensor, and the preparation of glass optical fibers by chemical etching. In addition, the description of the important technical components (lasers, filter and so on) for the SNOM experiment concludes this chapter. The third chapter focus is on the time-gated pulse excitation and optical signal detection of SNOM images. The technical configuration of the SNOM is presented in more details. The fourth chapter starts from with a broad overview of living bacteria Escherichia coli (E.coli) and of the Green Synechococcus – type of Picocyanobacteria cell lines, and presents the distinctive advantages for of the SNOM investigations working in native physiological conditions. The following sections emphasize our obtained high-resolution experimental SNOM result measurements, then followed a discussion of the results follows. The sample preparation and deposition procedures of the living cells for the scanning will are also discussed in this chapter. The chapter five introduces our recently developed SNOM sensor, based on polymethylmethacrylate (PMMA) plastic optical fiber. The chemical preparation of sharp (>100 nm) tips, and its significant advantages will be illustrated by experimental results.

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