Spectrally Resolved Fluorescence Lifetime Imaging Microscopy
Authors: Hanley, Quentin S.; Arndt-Jovin, Donna J.; Jovin, Thomas M.
Source: Applied Spectroscopy, Volume 56, Issue 2, Pages 50A-69A and 145-274 (February 2002) , pp. 155-166(12)
Publisher: Society for Applied Spectroscopy
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Abstract:
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.Keywords: S MATRIXES; LCD; SPATIAL ENCODING; IMAGING SPECTROSCOPY; SPECTRAL IMAGING; MICROSCOPY; HADAMARD TRANSFORMS; FLUORESCENCE LIFETIMES; EGFP; PROPIDIUM IODIDE; FLUORESCENT PROTEINS; FREQUENCY DOMAIN; GLOBAL ANALYSIS; FLUORESCENCE LIFETIME IMAGING MICROSCOPY; FLIM; FLUORESCENCE RESONANCE ENERGY TRANSFER; FRET
Document Type: Research article
DOI: 10.1366/0003702021954610
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