FlatFLIM: enhancing the dynamic range of frequency domain FLIM

Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.