Phasor deconvolution on directly sampled pulse for high-resolution and high-speed fluorescence lifetime imaging microscopy

Recently, phasor approach has emerged as a powerful tool for extracting fluorescence lifetime and has been utilized as a biochemical component analyzing tool without complicated fitting algorithms. In this study, we propose the new method to obtain phasors from directly sampled waveforms. With deconvolution using optically obtained instrumental response function (IRF), fluorescence lifetime can be successfully measured with high precision (~ 40 psec). Cells under the various metabolic conditions were imaged through label-free fluorescence lifetime imaging microscopy with targeting nicotinamide adenine dinucleotide (NADH) and their phasors exhibited distinct clusters on phasor plots corresponding to different culturing conditions.