Development and application of photon counting techniques for fluorescence microscopy

• Main Author: Zanda, Gianmarco
• Other Authors: Ameer-Beg, Simon ; Suhling, Klaus
• Published: King's College London (University of London) 2016
• Subjects: 530
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.679784

Fluorescence lifetime imaging microscopy (FLIM) is a key technique to image cells as, in addition to the advantages of standard fluorescence microscopy, it allows to study the environment and probe interaction in living specimens. Implementing FLIM via Single Photon Counting (SPC) proved to be the most effective technique considering the fluorophores limited photon budget before being irreversibly bleached. This thesis focuses on the development and application of Single Photon Counting techniques to imaging systems and to spectroscopy. Firstly, the BODIPY-C12 molecular rotor was used to determine dyeconcentrations between 3 and 16 μM for living cells and lipid droplets via FLIM and intensity measurements A novel compound, named ET, was tested for the first time on living cells and its possible applications as a molecular rotor discussed. The use of an Electron Bombarded Charge-Coupled Device (EBCCD) camera as a parallel-processing Time to Amplitude Converter device for SPC Imaging with sub-frame exposure time resolution was investigated and, although not implemented, the results supports the proposed method. In order to design a wide field time-correlated single photon counting system, a Complementary Metal-Oxide Semiconductor (CMOS) Fast- Camera was coupled with an 3-stage image intensifier. This proved to be suitable for luminescence lifetime measurements of a Ruthenium complex, with results of 1.7μs comparable with confocal scanning Time-Correlated SPC (TCSPC). Finally, conclusions and future work are discussed.