Deep insights from 3D FLIM: metabolic FLIM analysis taken to the 3rd dimension

Fluorescence lifetime imaging (FLIM) is a versatile tool, even detecting cancers without staining agent from NADH and FAD autofluorescence. While confocal techniques provide wonderful images of 2D structures, they risk neglecting crucial details in the out-of-image-plane dimension. Thorough understanding of three-dimensional samples is obtained through "Z-stack measurements", coupled with appropriate functional sectioning capabilities. Here, we demonstrate the combination of Becker&Hickl's advanced SPCimage NG data analysis suite with an easy-to-follow ImageJ/Fiji 3D rendering workflow to generate volume FLIM images of real samples. Combined with Becker&Hickl's renowned lifetime measurement precision 3D FLIM elevates metabolic imaging and deep-tissue work.