This digital resource is a scientific article from the Journal of Microscopy, providing a comprehensive overview of Fluorescence Lifetime Imaging (FLIM) techniques and their applications. FLIM is an advanced microscopy technique that measures the excited-state lifetime of fluorophores, which is independent of fluorophore concentration, photobleaching, or excitation intensity. This characteristic makes FLIM a robust tool for quantitative biological measurements. The article delves into the technical aspects of FLIM, covering various approaches such as time-domain FLIM (e.g., time-correlated single photon counting, TCSPC) and frequency-domain FLIM. It explains how these methods measure the decay rate of fluorescence after excitation, providing insights into the fluorophore's molecular environment. The technical part focuses on the instrumentation and methodologies used to acquire and analyze fluorescence lifetime data. Key applications discussed include the measurement of molecular environment parameters (e.g., pH, ion concentrations), protein-interaction studies via Förster Resonance Energy Transfer (FRET), and metabolic state analysis of cells and tissues through autofluorescence. FLIM-FRET, for instance, offers advantages over intensity-based FRET by being independent of donor concentration and avoiding issues like donor bleed-through. This article is invaluable for researchers in cell biology, biochemistry, and biophysics who require quantitative information beyond simple intensity measurements. It guides them in understanding how FLIM can provide deeper insights into cellular processes, protein-protein interactions, and physiological changes, making it a powerful tool for advanced biological research. The detailed discussion of techniques and applications helps researchers implement FLIM effectively and interpret their results accurately.