TY - THES ID - 137881736 TI - Phasor-based fluorescence lifetime imaging microscopy of single HIV-1 particles AU - Rodríguez Martín, Laura AU - Hendrix, Jelle AU - Hofkens, Johan. AU - KU Leuven. Faculteit Bio-ingenieurswetenschappen. Opleiding Master of Bioinformatics (Leuven) PY - 2016 PB - Leuven KU Leuven. Faculteit Bio-ingenieurswetenschappen DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:137881736 AB - Employing lifetime analysis together with single-molecule detection techniques opens the door for studying and monitoring protein-protein interactions inside viruses and with cellular structures in real time. It would allow for the detection of FRET during the whole viral infection cycle without the need of photobleaching, which permits the detection of fluorescent proteins using FRET more than once facilitating the real-life tracking of all the interactions. In the case of HIV-1, interactions of integrase (IN) are of special interest as it is known to have an important role for viral DNA integration in the host cell. Throughout this thesis, a method has been developed to perform lifetime analysis of IN attached to eGFP inside an HIV-1 virus. Using the software PAM, lifetimes of eGFP inside HIV-1 viruses in different conditions were obtained using phasor approach and comparing them with the lifetimes obtained utilizing already used methods in the time domain to test in which condition the phasor approach performs better and obtains a correct lifetime. Results show that phasor approach gives an accurate lifetime avoiding the problems of time domain analysis, such as time consuming exponential fitting. Furthermore, it is shown that a photon count of at least 1,000 photons per pixel is necessary. ER -