This dissertation revolves around the design and implementation of novel instrumentation and related measurement techniques, at the single molecule level, for use in biophysical research. Chapter 1... Show moreThis dissertation revolves around the design and implementation of novel instrumentation and related measurement techniques, at the single molecule level, for use in biophysical research. Chapter 1 presents an introduction to the field of fluorescence-based single molecule measurements. In particular, two fluorescence-based confocal microscopy developments are presented in Chapter 2 and Chapter 3, respectively. The room-temperature microscope in chapter 2 combines single-molecule detection capability, together with simultaneous polarization, fluorescence correlation spectroscopy, and lifetimes decay measurements with picosecond resolution. Its associated advanced electronics is also presented and discussed. On the other hand, Chapter 3 presents the novel thermal-cycles confocal microscope, capable of exerting extreme thermal gradients on single biomolecules for exploring their out-of-equilibrium configurations. The excitation and thermal pulses generation is achieved by purely optical means. To the author of this dissertation´s best of knowledge, up to date this system designed here is unique in its type in the world. Finally, Chapter 4 presents an experimental test of a prediction made by Theodor Förster in 1949, regarding the non-exponential lifetime decay for a single donor molecule surrounded by an ensemble of acceptors, and its comparison at the single-molecule level. Rhodamine 6G is used as the donor, and embedded in PMMA matrix. Show less
