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

James A. Levitt; Pei-Hua Chung; Klaus Suhling

doi:10.1117/1.JBO.20.9.096002

3 September 2015 Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

James A. Levitt, Pei-Hua Chung, Klaus Suhling

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Journal of Biomedical Optics, Vol. 20, Issue 9, 096002 (September 2015). https://doi.org/10.1117/1.JBO.20.9.096002

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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James A. Levitt, Pei-Hua Chung, and Klaus Suhling "Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity," Journal of Biomedical Optics 20(9), 096002 (3 September 2015). https://doi.org/10.1117/1.JBO.20.9.096002

Published: 3 September 2015

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