Spectrally resolved fluorescent lifetime imaging

Placing an imaging spectrograph or related components capable of generating a spectrum between a microscope and the image intensifier of a conventional fluorescence lifetime imaging (FLIM) system creates a spectrally resolved FLIM (SFLIM). This arrangement provides a number of opportunities not readily available to conventional systems using bandpass filters. The examples include: simultaneous viewingofmultiple fluorophores; tracking of both thedonor and acceptor; and observation of a range of spectroscopic changes invisible to the conventional FLIM systems. In the frequency-domain implementation of the method, variation in the fractional contributions fromdifferent fluorophores along the wavelength dimension can behave as a surrogate for a frequency sweep or spatial variations while analysing fluorophore mixtures. This paper reviews the development of the SFLIMmethod, provides a theoretical and practical overview of frequency-domain SFLIM including: presentation of the data; manifestations of energy transfer; observationofmultiple fluorophores; and the limits of single frequencymethods.