Synthesis and characterization of dye-doped nanoparticles for use in super-resolution fluorescence microscopy

Super-resolution optical microscopy can be divided into two categories: single molecule localization methods such as stochastic optical reconstruction microscopy (STORM), and patterned excitation methods such as stimulated emission depletion (STED). These techniques were created due to the need to surpass the limitations of wide-field optical microscopy, specifically the optical diffraction limit or Abbe Diffraction Limit, which states that the smallest resolvable distance is ½ the excitation wavelength. This places limits on what can be studied, as many structures and processes in living systems are not able to be resolved due to their size; while such small structures can be studied using techniques such as transmission electron microscopy, such techniques cannot provide information on living systems as sample preparation involves ending life processes of the sample. There are very specific requirements for the fluorophores used for super-resolution techniques, the most important of which is the ability to photoswitch, or switch from a bright state to a dark state. This work focuses on the use of photoblinking dye-doped nanoparticles as fluorescent probes for localization-based super-resolution microscopy. Fluorescence techniques and super-resolution microscopy are discussed in greater detail in Chapter I and the use of nanomaterials as super-resolution fluorescent probes are discussed in Chapter II. Chapters III, IV, V, and VI discuss the work done on developing fluorescent nanoparticle probes for localization-based super-resolution microscopy. Their fluorescence and photoblinking behavior were assessed under different excitation wavelengths and in different environmental conditions and were determined to be suitable for use in localization-based super-resolution microscopy.

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