Fluorescence lifetime imaging comes of age how to do it and how to interpret it.

Fluorescence lifetime imaging (FLI) has been used widely for measuring biomedical samples. Practical guidelines on taking successful FLI data are provided to avoid common errors that arise during the measurement. Several methods for analyzing and interpreting FLI results are also introduced; e.g., a model-free data analysis method called the polar plot allows visualization and analysis of FLI data without iterative fitting, and an image denoising algorithm called variance-stabilizing-transform TI Haar helps to elucidate the information of a complex biomedical sample. The instrument considerations and data analysis of Spectral-FLI are also discussed.