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.

Scanning near-field optical microscopy with new probes and feedback modes

Anton Smirnov

Scanning Near-field Optical Microscopy (SNOM) technique enables to overcome Abbe diffraction limit of far-field optics as well as to obtain simultaneously optical and topographical images. While the optical resolution of the method is limited by the aperture size and is typically 50 - 100 nm, an excellent spatial resolution in a topography channel can be realized. Naturally, we need a convenient and precise method to control the distance between the tip and sample for the successful operation of any SNOM device. Nowadays, the most popular method of the SNOM tip-sample distance control is the shear force - based feedback employing a glass fiber attached to the quartz tuning fork (TF). However, the shear-force distance control method is far from the ideal one. The crosstalk between optical and topographical image can warp the results. The forces between the tip and sample are high and in many cases might be destructive. We report the realization of a new approach to the problem: bent sharpened glass optical fibers with carefully controlled sizes of the bent part and the radius of the curvature of the bending were prepared and experimentally exploited as SNOM probes. Detailed analysis of fiber vibration modes shows that realization of truly tapping mode of the probe dithering requires extreme caution. In case of using the second resonance, mode probes vibrate mostly in the shear-force mode unless the bending radius is rather small (0.3 mm) and the probe's tip is short. The probes having these characteristics were prepared and attached to the TF in the double resonance conditions, which enables to achieve a significant quality factor of the sensor. Another common problem of most aperture SNOMs is the fragility of the tip. We proposed and realized the use of different plastic fibers to solve this problem. These fibers look very promising for the use as SNOM probes and are characterized by much less fragility (compare glass and plastic) and greater ease of the tip preparation. For such preparation, hazardous treating with hydrofluoric acid, which remains the most popular approach to prepare SNOM probes from the glass fibers, can be entirely avoided. Fluorescence Resonance Energy Transfer (FRET) is one of the most promising ways to improve the spatial resolution of the SNOM, and the central part of the Thesis is devoted to the elaboration of FRET SNOM. The idea is to use a donor (acceptor) nanoparticle/molecule as local fluorescence center attached to the tip and measure the fluorescence induced by it in the sample (or vice versa) due to the FRET. Ten years ago, this idea was realized at the single molecule level with CdSe nanocrystals and appropriate dye molecules. Despite the high spatial resolution (better than 20 nm) attained in this experiment, it remains an isolated one, and this is for a valid reason: albeit rather large, the photostability of dye molecules and semiconductor nanocrystals still enables to use a single fluorescence center exploiting for imaging only a few minutes at best. Fluorescent centers with high photostability should be used to overcome this problem. Earlier, claimed to be very photostable and bright NV color centers in nanodiamond crystals were proposed. In the Thesis, we show that such a system is not suitable to realize a single fluorescent center FRET SNOM method. We propose to use specific rare-earth ions in crystals to achieve the goal, in particular, LuBO3(Tb) micro- and nanocrystals.

EPFL2019

Chemically etched plastic optical fiber probe for near-field scanning optical microscopy in liquids

Giovanni Dietler, Kanat Dukenbayev, Anton Smirnov

Some preliminary results and experimental details of Near-field Scanning Optical Microscopy (NSOM) operation in liquids have been reported by us earlier(1-4). Here we present the first use of custom made polystyrene/poly (methyl methacrylate) (PMMA) optical fibers to assemble new NSOM probe/sensor for operation in liquids. Assembled NSOM probe has quite large quality factor Q ranging 2000-6000 in air(1), and 300-900 when immersed 0.2-0.3 mm deep into the water. Such montage demonstrates high mechanical durability permitting to scan different samples during many hours or even days, and overall low cost in comparison with NSOM probes based on glass optical fibers. A specially prepared optical fiber with 125 mu m diameter (from Paradigm Optics Company, USA) (polystyrene core diameter is 0.85 mu m, n(core) = 1.59 and PMMA cladding, n(core) = 1.49) was chemically etched using a 9: 1 mixture of dichloromethane and ethyl acetate(2). As result of the etching, a smooth and sharp tip is formed with a typical radius of the curvature equal to 50 - 170 nm. For completeness, earlier unpublished images of living Picocyanobacteria bacteria obtained using glass fiber-made NSOM probes are also presented.

SPIE-INT SOC OPTICAL ENGINEERING2019

Scanning near-field infrared microscopy and spectromicroscopy applied to nano-systems and cells

Dusan Vobornik

This thesis further explores the possibilities of scanning near-field optical microscopy (SNOM) in both materials and life sciences. Two experimental SNOM setups were developed: one designed for infrared spectroscopy applications and the other for the imaging of fluorescently labeled samples. The results of the experiments that were conducted with both setups are presented and analyzed in this thesis. Diffraction limits the resolution of lens-based microscopes to a value close to that of the wavelength of the light that is used to illuminate the samples. SNOM instruments overcome this limit by probing the near-field light — the light that remains within close vicinity of the sample and decays exponentially away from it. The SNOM concept and instrumental design are described. A comparison of SNOM with other novel microscopies (electron, atomic force, and scanning tunneling microscopies) outlines the main advantages of SNOM — the sole optical microscopy technique with a nanometer resolution. Aperture-based infrared SNOM (IR-SNOM), performed in the spectroscopic mode using the Vanderbilt University free electron laser, recently started delivering spatially resolved information on the distribution of chemical species and on other laterally fluctuating properties. The IR-SNOM combines the IR spectroscopy's chemical specificity and the SNOM's high optical and topographical resolutions. The IR-SNOM experimental setup is described in detail and results from the study of cells, boron nitride and lithium fluoride thin films are presented and analyzed. They demonstrate the great potential of this new technique. An overview of the development of IR-SNOM, as well as a consideration of its future possibilities are presented. The newly built SNOM instrument at EPFL was used for experiments that are complementary to the aforementioned IR-SNOM experiments. Specifically, the new SNOM enabled the study of fluorescently marked samples. Images of fluorescently labeled cells and carbon nanotubes are presented and analyzed. The results further demonstrate the exciting possibilities of SNOM.