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

Journal of Biomedical Optics
James A LevittKlaus Suhling

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 ma...Continue Reading

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Citations

May 3, 2016·Sensors·Liisa M Hirvonen, Klaus Suhling
Nov 16, 2019·Beilstein Journal of Organic Chemistry·Aydan YadigarliHolger Schönherr
Oct 21, 2020·Proceedings of the National Academy of Sciences of the United States of America·Clinton BelottMichael A Menze
May 8, 2021·Journal of Cell Science·Ruslan I DmitrievMargarida M Barroso
May 25, 2021·Frontiers in Molecular Biosciences·Massimiliano GalluzziJiangyu Li

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