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Applied Spectroscopy

Applied Spectroscopy


  • Vol. 56, Iss. 2 — Feb. 1, 2002
  • pp: 155–166

Spectrally Resolved Fluorescence Lifetime Imaging Microscopy

Quentin S. Hanley, Donna J. Arndt-Jovin, and Thomas M. Jovin

Applied Spectroscopy, Vol. 56, Issue 2, pp. 155-166 (2002)

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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.

Quentin S. Hanley, Donna J. Arndt-Jovin, and Thomas M. Jovin, "Spectrally Resolved Fluorescence Lifetime Imaging Microscopy," Appl. Spectrosc. 56, 155-166 (2002)

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