This thesis describes the development of a tip-enhanced fluorescence microscope, based upon combining confocal fluorescence methods with a commercially-available atomic force microscope (AFM).
A microscope which is capable of simultaneous fluorescence and atomic force measurements has been realised. This has been achieved by mounting an AFM onto an inverted optical microscope, allowing the sample to be fluorescently excited from underneath. The incident position of the excitation spot at the sample can be controlled by a 2D galvanometer present in the excitation beam path. A program has been developed which aligns the laser spot with the AFM-tip by correlating sample features, and then applying a corresponding voltage to the galvanometer, in order to translate the beam towards the AFM-tip. A result of this process is that the fields-of-view of the fluorescence and atomic force images are also aligned, enabling simultaneous correlative microscopy. The benefits of performing these techniques simultaneously include eliminating the need to image over large areas with both techniques to ensure overlapping fields-of-view (as would be required if these measurements were being performed consecutively). It also unlocks the potential to track the mechanical changes of a known structure over time. Moreover, elements of the alignment program may be useful for general applications in correlative microscopy, providing an automated tool for overlaying images which have been obtained using different techniques.
Alongside simultaneous imaging, this alignment method is also a potential technique for introducing a tip-enhancement effect into this particular instrument. A method has been developed which acquires data from a single photon counting module (SPCM) and the AFM using a LabVIEW field programmable gate array (FPGA), and subsequently correlates the number of detected photons from the sample with the position of the AFM-tip at that time. Although fluorescence enhancement was not detected using this instrument, the method was sufficient enough to detect scatter as the AFM-tip made contact with the sample; this is evidence that the method can successfully measure the number of photons detected with respect to tip-sample separation and should be able to also detect an increase in photons due to enhancement, if the effect can be optimised
