Fluorescence Lifetime Imaging Microscopy

Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay characteristics of endogenous fluorophores, providing quantitative insights into biological systems. This review examines two complementary analytical approaches: multi-exponential fitting and phasor plot representation. Multi-exponential fitting mathematically decomposes fluorescence decay curves to characterize lifetime components, enabling the quantitative assessment of cellular metabolism through analysis of NAD(P)H, FAD, tryptophan, and collagen. The phasor plot approach transforms lifetime data into a two-dimensional coordinate system through Fourier analysis, offering intuitive visualization of multi-component systems. Clinical applications span dermatology, oncology, and metabolic research, with FLIM differentiating skin cell types, monitoring wound healing, and enabling cancer diagnostics through exosome analysis. Both approaches offer complementary strengths: multi-exponential fitting provides quantitative parameters for comparative studies, while phasor plots enable rapid visual discrimination of biological states.