Conventional fluorescence images show the spatial distribution of emission intensity. These kinds of images contain further information that is concealed in intensity images.
To unveil this information, the emission is separated by the lifetime of the excited state. This approach is called "Fluorescence Lifetime Imaging" (FLIM) or "τ-mapping", a technique that can reveal differences which are indis-cernible with conventional fluorescence microscopy. Such differences are caused by the molecular environment of the fluorescent dye, for example pH, polarity, or other molecular compo-nents. Furthermore, the FLIM-signal is more suitable for quantitative measurements: the results are in many cases more precise and reproducible than customary intensity measurements. It is the ideal method for modern FRET-Biosensors which are used to measure, for example, Ca2+ or cAMP concentrations in living cells.
Until lately, procedures for recording FLIM images were quite complex and cumbersome and only a small circle of specialists met that challenge. In addi-tion, recordings were limited to low frame rates, prohibiting sensible research on living material. Recently, new technologies and data evaluation procedures changed that picture. Frame rates are at least 10 times faster and the demands of operating such a system are not substantially different from recording classical confocal images.
This article outlines color-coding of intensity versus lifetime images and how the combination of the two contrast methods can reveal new insights, even within narrow spectral bands.