Fast algorithms for the analysis of spectral FLIM data

The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM) allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and degree of binding. Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to condense the inherent information to answer the scientific questions in a straightforward way. Different analysis techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination of environmental parameters and distance measurements via FLIM FRET. In order to reach highest sensitivity in single photon detection, different detector types are investigated and developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels. High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.