| Description |
This dissertation describes the advancements made towards the implementation of Tip-Enhanced Fluorescence Microscopy (TEFM) in imaging biological specimens. This specialized type of microscopy combines the chemical specifi city of optical microscopy techniques with the resolution of atomic force microscopy (AFM). When an AFM probe is centered in the focal spot of an excitation laser with axial polarization, the probe concentrates the optical field such that it can be used to induce nanometer scale fluorescence. The physical mechanisms of this optical field enhancement are set forth in detail. The feasibility of this technique for imaging bimolecular networks is discussed in regard to the requirements for adequate image contrast, as well as for obtaining fi eld enhancement in aqueous environments. A semianalytical model for image contrast for TEFM has been developed. This model shows that using demodulation techniques greatly increases the image contrast attainable with this technique, and is capable of predicting the requisite enhancement factors to achieve imaging of biomolecular networks at good contrast levels. This model predicts that signal enhancement factors on the order of 20 are needed to image densely packed samples. This dissertation also highlights a novel tomographical imaging approach. By timestamping the fluorescence photon arrival times, and subsequently correlating them to the timestamped motion of a vertically oscillating probe, a three-dimensional map of tip-sample interactions can be constructed. The culmination of these advancements has led to the ability to map the interactions between single carbon nanotubes and single fluorescent nanocrystals (quantum dots). Various attempts at using TEFM in water have been thus far unsuccessful. Several explanations for this shortfall have been identi ed|understanding these shortcomings has helped to identify the optimal excitation conditions for field enhancement. |
