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Single molecule fluorescence spectroscopy has attracted considerable attention over the past two decades. Measurement on a single entity provides an opportunity to avoid ensemble averaging which is always present in conventional bulk fluorescence measurements. This makes single molecule spectroscopy particularly interesting for biophysics and biochemistry where heterogeneous systems are often encountered. The general interest of this thesis is in studies of single immobilised molecules carried out at room temperature. One of t.he major issues of single molecule spectroscopy is finding a suitable immobilising medium. Inorganic silica matrices prepared by the sol-gel method have a great potential to provide a close- to-natural immobilising environment even for sensitive biomolecules and thus allow investigation of their natural behaviour on the most fundamental level. In order to be able to tailor both physical and chemical properties of the final gel, it is of great importance to develop reliable methods to control each stage of polymerisation. In one part of this thesis, applications of fluorescent probes to investigation of sol-gels properties, as well as monitoring the gel assembly process, are discussed. The thesis further presents studies of the genetically engineered glucose binding protein labelled with the environmentally sensitive dye badan. This system was developed in a search for an appropriate recognition-reporter unit to serve as a part of fluorescence-based sensor for continuous blood glucose monitoring. This labelled biomolecule represents an interesting subject for a single molecule study. Due to technical reasons however, single molecule spectroscopy could not be applied in this case. Therefore, conventional ensemble fluorescence spectroscopy methods were used to characterise behaviour of the labelled protein at different glucose concentrations. The last part of the thesis deals with instrumental aspects of single molecule imaging and spectroscopy. The aim of the work was to assess the applicability of a freshly installed commercial microscope a-SNOM (WITec GmbH) in single molecule fluorescence studies and at the same time to adopt the technique for future experiments in our research group.

A novel approach to absorption spectroscopy is presented which achieves, simultaneously, wide spectral coverage and high spectral resolution. The principle of the technique - dubbed multi-mode absorption spectroscopy (MUMAS) - is described, and demonstrations of the principle are reported using two multi-mode sources: diode lasers and micro-cavity solid state lasers. The technique is shown to have potential for the detection of multiple species and multiple parameters using a single laser and a single detector.