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Fluorescence lifetime imaging is a very powerful tool for the determination of changes in the environment of the fluorophores under study. Among these changes, the decrease in lifetime that the presence of a quencher (like a FRET acceptor) induces is of paramount interest. Changes in fluorescence lifetime can be used to measure conformational changes, differences in pH, calcium concentration… In our case we have decided to use the FRET pair formed by mTFP1 and mVenus to monitor the oligomerization state of HIV-1 integrase by alternatively labelling the integrase with mTFP1 or mVenus. The first step of our study was to assemble the confocal microscope in which we were going to image the different viral fusion constructs. Although we only needed two excitation lines and two detection channels we decided to build a setup with 6 excitation lines and 4-color detection as this would provide with more flexibility for future research. Once the physical assembly of the microscope was done we used fluorescence correlation spectroscopy to test the alignment of all the components of the setup. FCS analysis of free dyes confirmed that the microscope had been well assembled. Additionally, we used spatial autocorrelation to determine accurately the pixel size of the images recorded. Fluorescence lifetime imaging is usually analysed by fitting the measured decays of the fluorophores. The main drawback fitting methods have is that multiexponential decays are difficult to analyse properly and expertise is required to avoid artefacts. Therefore, we decided to use the phasor approach for fluorescence lifetime imaging analysis. This approach eliminates the need for fitting steps simplifying data analysis. Our results show that phasor approach is a valid analysis tool for the determination of fluorescence lifetime. The second step was to develop the required Matlab routines that allowed for single particle selection in an image and to calculate the phasors of those particles. Initial tests were performed using viral particles labelled with eGFP and confirmed that the developed code and phasor approach also work well for single particle analysis. The lifetime analysis of integrase labelled with mTFP1 and mVenus revealed an unexpected increase in the lifetimes of the two fluorescent proteins in the fusion construct compared with the free protein. To test if this effect was a result of an increase in the rigidity of the fluorescent protein when they were fused to the integrase we studied the fluorescence anisotropy of the free protein and labelled viruses in solution. No apparent changes in fluorescence anisotropy were observed although further experiments are still required to determine the origin of this increase in fluorescence lifetime. Our final aim, was to determine if HIV particles localized in different points inside the cells would show differences in FRET efficiency. To do so we infected HeLa cells with integrase-labelled HIV and imaged them. No conclusion can be drawn yet from this experiments and further research is needed to accomplish the final goal of this project.