Fluorescence Lifetime Imaging (FLIM) SymPhoTime 64 supports analysis of images with dimensions up to 4096 x 4096 pixels. As a first analysis step, a special “fast FLIM” procedure can be applied that yields immediate results and is therefore very useful for a quick preview, assessment of the image quality or the selection of regions-of-interest (ROIs) for more detailed analysis. ROIs are especially useful if an analysis procedure should be restricted to certain areas of the sample such as e.g. the cell nucleus, the cell membrane or microcrystalline substructures. A detailed FLIM analysis is then based on fitting an exponential decay function to the acquired fluorescence decay in each image pixel. In that way, the SymPhoTime 64 software permits to extract up to five different lifetimes and related amplitudes from the measured data.
Even the finite temporal resolution of the system (Instrument Response Function, IRF) can be corrected using a dedicated numerical reconvolution algorithm with either measured data or individually calculated correction profiles.
Förster Resonance Energy Transfer (FRET) FLIM-FRET measurements can be evaluated using either the fluorescence intensity or the fluorescence lifetime as parameter. In the latter procedure, a dedicated fitting function is applied. FLIM-FRET analysis results can be directly visualized in a false color representation. The FRET efficiency histogram and the histogram of the donor-acceptor distances in units of the Förster distance are always calculated.