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

TY - JOUR

T1 - Spectrally resolved fluorescence lifetime imaging of Nile red for measurements of intracellular polarity

AU - Levitt, James A.

AU - Chung, Pei Hua

AU - Suhling, Klaus

PY - 2015/9/1

Y1 - 2015/9/1

N2 - 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.

AB - 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.

KW - HeLa cells

KW - lipid droplets

KW - Nile red

KW - phasor analysis

KW - spectrally resolved fluorescence lifetime imaging microscopy

KW - time-correlated single photon counting

UR - http://www.scopus.com/inward/record.url?scp=84942645773&partnerID=8YFLogxK

U2 - 10.1117/1.JBO.20.9.096002

DO - 10.1117/1.JBO.20.9.096002

M3 - Article

AN - SCOPUS:84942645773

SN - 1083-3668

VL - 20

JO - Journal of Biomedical Optics

JF - Journal of Biomedical Optics

IS - 9

M1 - 096002

ER -