Many biological samples exhibit autofluorescence. Its often broad spectra can interfere with fluorescent labelling strategies.
However, autofluorescence as such has its merits, too. It is an intrinsic form of fluorescent label which comes for free and serves as completely non-invasive contrast.
Even more, the combination of spectral imaging with fluorescence lifetime information is especially useful to distinguish and potentially identify different fluorescent species in biological samples. An automated acquisition of the underlying FLIM λ stacks can be easily done within the FLIM wizard using SP FLIM detectors.
- Lambda-Stacks of FLIM images of invading pathogenic hyphae in tomato fruit (1) using autofluorescence excited with 470 nm.
- Spectral scan of 50 nm steps from 440 to 740 nm (2).
- Intensity spectra (3) and lifetime spectra (4) show strongly overlapping, non-separable species.
- Spectral information with fluorescence lifetimes allows disentangling a complex mixture of autofluorescent species (i.e. “fingerprinting” of each species).