FLIM (Fluorescence Lifetime Imaging Microscopy) represents a new paradigm in laser scanning fluorescence microscopy, enabling the measurement not only of fluorescence intensity and color, but also of the average fluorescence decay time (known as fluorescence lifetime) within both biological and non-biological samples.

In addition to the spatial and structural information (imaging) provided by laser scanning microscopes, FLIM technology delivers functional insights into the sample. The fluorescence lifetime of fluorophores is influenced by changes in the cellular microenvironment; therefore, FLIM data can be correlated with various molecular properties of the subcellular environment, as well as with parameters such as concentration, pH, and/or the presence of specific molecular interactions.

The fifth edition of the Microscopy School focused on understanding the optical principles and applications of FLIM microscopy, combined with standard imaging techniques such as confocal microscopy, to achieve improvements in spatial and temporal resolution. This approach enables detailed subcellular imaging with molecular specificity.