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 <i>Drosophila</i> 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 <i>Drosophila</i> salivary gland explants exhibited strong fluorescence resonance energy transfer (FRET), a result consistent with the known nucleosomal structure of eukaryotic chromatin.
Quentin S. Hanley, Donna J. Arndt-Jovin, and Thomas M. Jovin, "Spectrally Resolved Fluorescence Lifetime Imaging Microscopy," Appl. Spectrosc. 56, 155-166 (2002)
References are not available for this paper.
OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.