Research Papers: Imaging

Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

[+] Author Affiliations
James A. Levitt, Pei-Hua Chung, Klaus Suhling

King’s College London, Department of Physics, Strand, London WC2R 2LS, United Kingdom

J. Biomed. Opt. 20(9), 096002 (Sep 03, 2015). doi:10.1117/1.JBO.20.9.096002
History: Received January 20, 2015; Accepted July 17, 2015
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Abstract.  Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ϵ, is around 5 in lipid droplets and 25<ϵ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.

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© 2015 Society of Photo-Optical Instrumentation Engineers

Citation

James A. Levitt ; Pei-Hua Chung and Klaus Suhling
"Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity", J. Biomed. Opt. 20(9), 096002 (Sep 03, 2015). ; http://dx.doi.org/10.1117/1.JBO.20.9.096002


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