| Summary: | A range of fluorescence fluctuation analysis methods were developed and applied to labels commonly used in biological samples. Various super-resolution techniques were demonstrated in vitro and in vivo using objective-type Total Internal Reflection Fluorescence Microscopy (TIRFM). An experimental Fluorescence Correlation Spectroscopy (FCS) setup was developed along with associated post-acquisition data algorithms. The technique was applied to investigate the stoichiometry of a protein subunit of ribonucleotide reductase (RNR). FCS method was adapted with an Electron Multiplying Charge Coupled Device (EMMCD) detection scheme and applied to inorganic Quantum Dots (QDs) diffusing in solutions of different viscosities. Super-resolution Optical Fluctuation Imaging (SOFI) algorithm was implemented in ImageJ software and conclusive results obtained on reference samples of QDs and combed DNA. Potential applications of temporal correlation analysis to the study of diffusive processes and single particle tracking were also discussed. A new super-resolution technique applicable to multiple adjacent fluorescent molecules called Direct Object Resolution by Image Subtraction (DORIS) was developed and tested with QD complexes. The method enables one to accurately map the position of two emitters displaying intermittency in their fluorescence emission and separated by a distance below the diffraction limit, without the need of complex instrumentation or analysis. The technique relies on the subtraction of the Point Spread Function (PSF) of each single fluorescent probe, and is in theory applicable to any blinking or flickering dye. The principle was first demonstrated on simulated data and experimentally on QDs coupled by 100-basepair double-stranded DNA constructs. Super-resolution by image subtraction was further applied in vivo in S. pombe cells, where distances between clustered fluorescent fusion proteins were accurately determined. The selective activation of photoswitchable probes mEos3 was exploited to optimise the DORIS subtraction process and provided a simple method to determine the relative positions of closely spaced emitters within an aggregate, as encountered in association sites or multimeric complexes.
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