| dc.description.abstracteng | Super-resolution far-field fluorescence microscopy (optical nanoscopy) is a mature set of methods which enable visualization of the nanometer-scale distribution of objects such as organic molecules, photoswitchable proteins, point-like defects in the diamond lattice, upconversion nanoparticles, semiconductor quantum dots, etc. Utilization of internal emitter states in the imaging scheme has made it possible to create contrast at the nanoscale in conventional lens-based optical imaging systems. Spatial resolutions down to the single nanometer scale have been reported in some cases. Nonetheless, routine biological experiments, which employ the molecular probes in physiological conditions, are hampered by photodestructive chemical reactions of fluorophores (photobleaching) which limit signal levels and the attainable resolution to ~20-50 nm. Substantial effort has been devoted to both finding more photostable markers and minimizing the light-induced damage by novel experimental strategies.
This thesis is concerned with coordinate-targeted super-resolution microscopy techniques applied to imaging of common molecular probes and semiconductor heterostructured nanowires at room temperature. These techniques have already demonstrated single nanometer resolution in far-field optical microscopy for ultra-stable emitters: namely color-defects in the diamond lattice. Similar resolution levels have not been reported for standard fluorophores imaged at room temperature. Therefore, the question arises how to increase the imaging capabilities of super-resolution microscopy under biologically relevant conditions.
The first aim of this thesis was to gain new insights into the photobleaching of organic dyes under photon fluxes typically applied in stimulated emission depletion (STED) microscopy. The impact of STED-light photons on the photobleaching of several organic molecules was studied with the goal to identify optimal imaging conditions. To this end, an optical system and experimental strategy were developed to systematically assess the key parameters in STED microscopy: transient de-excitation, irreversible photobleaching and STED-light-induced fluorescence resulting from undesirable excitation events caused by absorption of the STED-light photons. These parameters determine to what extent the STED concept works in practice with a specific dye. We varied the STED pulse duration from 0.13 ps to 500 ps and the time-averaged STED power up to 200 mW at 80 MHz repetition rate at the popular wavelength of 750 nm, examining common fluorescent compounds (ATTO590, STAR580, ATTO647N, STAR635P) in bulk experiments in thiodiglycol. The magnitude of photobleaching was different for different dyes. In general, two characteristic photobleaching regimes at a given STED pulse energy were found: intensity-dependent (high-order) and intensity-independent (low-order) bleaching. Surprisingly, for ATTO647N we observed a single effective photobleaching scaling over a wide range of STED peak powers (~0.1-200 W). Based on this observation, we developed an intuitive model for this dye which provides a quantitative prediction of the influence of STED-light photons on the resolution and bleaching. We inferred the spatial distribution of photobleaching probability, the role of detection time gating and the impact of residual STED intensity at the targeted coordinate on the resulting image at different STED pulse energies. The dominant bleaching mechanism determines
the optimal STED pulse duration to acquire a super-resolved image with minimal photodamage of the marker. High-order photobleaching can be efficiently reduced by increasing STED pulse duration up to roughly the fluorescence lifetime. For low-order photobleaching, chemical triplet quenchers and optical strategies allowing dark-state relaxation hold more promise. Overall, this is the first systematic
study of molecular photobleaching in STED microscopy, aimed at finding the optimal optical conditions which minimize STED-light-induced damage.
The second project within this thesis investigated the inherent photoluminescence of heterostructured gallium phosphide–gallium indium phosphide (GaP-GaInP) nanowires (NWs) to improve the resolution of far-field optical microscopy of these emitters. Due to their small diameter (<100 nm) but significantly larger length, and their tunable electro-optical properties, semiconductor nanowires are gaining interest in intra- and extracellular biological research. They hold potential as local probes of, for example, electric field or forces. Many of these applications require precise localization and identification of NWs featuring different geometries and surface coatings in a scattering biological environment. Traditional fluorescence microscopy, while suitable for nanowires and live-cell imaging, is hampered by limited spatial resolution. We addressed this issue and found that ground state depletion (GSD) microscopy can resolve heterostructured nanowires with a 5-fold resolution enhancement over confocal microscopy. This resolution improvement allowed us to image nanowires with diameters of 20-80 nm characterized by different geometries of photoluminescent GaInP segments of lengths 50-200 nm spaced by 50-150 nm. The influence of the GaInP segment sizes and positions within a single NW on the GSD image contrast is discussed in detail. The relative simplicity of this method and its moderate laser power requirements make it relevant for further biological studies. | de |