In fluorescence microscopy generally autofluorescence is viewed as a detrimental side-effect inherent to many biological samples. It tends to overlap with endogenous fluorescence labels sometimes masking its intensity. It can cause difficulties with spectral channel separation and with quantitation of intensities for ratiometric analysis. The often very broad emission spectra of autofluorescence can render it intractable by conventional spectral image recording.
However, autofluorescence as such has its merits, too. It is an intrinsic form of fluorescent label which comes for free and is completely non-invasive. The purpose of this application letter is to identify several autofluorescent components by means of fluorescence lifetime imaging microscopy (FLIM) and to demonstrate their utility in biomedical research.
Note: For a basic treatment of the FLIM techniquw please refer to the application letter FRET with Flim - Quantitative in-vivo Biochemistry
Most biological samples contain biochemical species which can give rise to autofluorescence. For example a cell’s redox potential is reflected in its concentration of NAD(P)H and flavins. The former are better excited with