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 ∼50 (λ/Δλ) in the current arrangement and a wavelength range of ∼430–750 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 ∼1 ns in buffer to ∼18 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.