Three-dimensional fluorescence lifetime imaging in confocal microscopy of living cells

Fluorescence lifetime imaging (FLIM) is a modern optical method which increases the potential of standard microscopy. This paper shows the possibilities of extended fluorescence lifetime evaluation and imaging in studying three-dimensional structures such as compartments of living cells with different fluorescence lifetimes. The method for quasi-FLIM image calculation is presented and image processing steps useful for biological experiments are suggested. The method was tested on isolated cardiomyocyte cells (CMs) and rat bone marrow stromal cells (MSCs) labelled with SPIO-rhodamine nanoparticles and stained with standard fluorescent dyes. We proved it is possible to use an exponential decrease of fluorescence in time and lifetime parameters for pseudo-colour 3D image mapping of living cells and their compartments that is not a standard function of confocal microscopes.