Reference-independent wide field fluorescence lifetime measurements using Frequency-Domain (FD) technique based on phase and amplitude crossing point.

Fluorescence lifetime imaging microscopy (FLIM) is an essential tool in many scientific fields such as biology and medicine thanks to the known advantages of the fluorescence lifetime (FLT) over the classical fluorescence intensity (FI). However, the frequency domain (FD) FLIM technique suffers from its strong dependence on the reference and its compliance to the sample. In this paper, we suggest a new way to calculate the FLT by using the crossing point (CRPO) between the modulation and phase FLTs measured over several light emitting diode (LED) DC currents values instead of either method alone. This new technique was validated by measuring homogeneous substances with known FLT, where the CRPO appears to be the optimal measuring point. Furthermore, the CRPO method was applied in heterogeneous samples. It was found that the CRPO in known mixed solutions is the weighted average of the used solutions. While measuring B16 and lymphocyte cells, the CRPO of the DAPI compound in single FLT regions was measured at 3.5 ± 0.06 ns and at 2.83 ± 0.07 ns, respectively, both of which match previous reports and multi-frequency analyses. This paper suggests the CRPO as a new method to extract the FLT in problematic cases such as high MCP gains and heterogeneous environments. In traditional FD FLIM measurements, the variation in phase angle and modulation are measured. By measuring over varying DC currents, another variation is detected in the FLT determined through the phase and modulation methods, with the CRPO indicating the true FLT.