Spectral decomposition of intracellular complex fluorescence using multiple-wavelength phase modulation lifetime determination: technical approach and preliminary applications.

Lifetime-based spectral decomposition using a frequency-domain phase/modulation technique is developed on a microspectrofluorimeter prototype. In a fluorescent mixture with strongly overlapping components, such measurements enable us to not only obtain excited state lifetimes of each fluorescent component but also determine the specific spectral contribution of each species without the use of any model spectra. Examples of such applications are first given for complex mixtures of highly overlapping fluorescent components in solution. Preliminary results concerning cellular applications are also reported. This allows us to follow the cellular uptake and intracellular stability of fluorescent labeled modified oligonucleotides in the context of antisense strategy studies. Indeed, the intracellular signal from the fluorescent label bound to oligonucleotides can be distinguished from those of the free label by its specific excited state lifetime.