Spectrally resolved fluorescence lifetime imaging microscopy

We report a system for collecting spectrally resolved fluorescent lifetime images. Frequency domain fluorescence lifetime detection was combined with two-dimensional spectral imaging in a programmable array microscope. The spectroscopic fluorescence lifetime imaging microscopy (sFLIM) system has a resolution of similar to50 (lambda/Deltalambda) in the current arrangement and a wavelength range of similar to430-750 nm. With the sFLIM system, we recorded the lifetime spectra of rhodamine 6G, rhodamine B, and the DNA intercalation dye propidium iodide (PI) in cuvettes and an EGFP-fusion of the histone 2A variant D protein in Drosophila salivary gland explants in the presence and absence of PI. In the absence of PI, the EGFP-fusion exhibited a lifetime of 2.7 ns with little variation in wavelength. The lifetime of PI alone ranged from similar to1 ns in buffer to similar to18 ns when intercalated in the nuclei of intact cells. The combination of EGFP and PI in the Drosophila salivary gland explants exhibited strong fluorescence resonance energy transfer (FRET), a result consistent with the known nucleosomal structure of eukaryotic chromatin.