Visualization of Lipid Droplets in Living Cells and Fatty Livers of Mice Based on the Fluorescence of π-Extended Coumarin Using Fluorescence Lifetime Imaging Microscopy

Abstract

Lipid droplets (LDs) are closely related to lipid metabolism in living cells and are highly associated with diverse diseases such as fatty liver, diabetes, and cancer. Herein we describe a π-extended fluorescent coumarin (PC6S) for visualizing LDs in living cells and in the tissues of living mice using confocal fluorescence lifetime imaging microscopy (FLIM). PC6S showed a large positive solvatochromic shift and high fluorescence quantum yield (>0.80) in both nonpolar and polar solvents. Additionally, the fluorescence lifetimes of PC6S were largely dependent on solvent polarity. The excellent spectral and photophysical properties of PC6S allowed its selective staining of LDs in living and fixed cells, and multicolor imaging. Fluorescence lifetime measurements of PC6S allowed estimation of the apparent polarity of LDs. The high photostability and long intracellular retention of PC6S supported in situ visualization of the formation processes of LDs resulting from the accumulation of fatty acid. Furthermore, intravenous administration of PC6S and use of the FLIM system allowed the imaging of LDs in hepatocytes in living normal mice and the growth of LDs resulting from the excess accumulation of lipids in high-fat-diet-fed mice (fatty liver model mice). Taking advantage of the high selectivity and sensitivity of PC6S for LDs in liver, we could visualize the adipocytes of lipid-rich tissues and LDs in kidney peritubular cells by PC6S fluorescence. These results demonstrated that PC6S combined with a FLIM system can be useful for monitoring and tracking the formation of LDs in both cultured cells and specific tissues and organs.

FLIM as a Promising Tool for Cancer Diagnosis and Treatment Monitoring

“…In addition to glucose consumption, Fig. 6 c also shows that a π-extended fluorescent coumarin (PC6S) combined with FLIM can be successfully applied for lipid droplet imaging related to lipid metabolism [ 109 ]. As glucose and lipid metabolism are both relevant to cancer cells’ activities, these strategies are promising for cancer diagnosis and anti-cancer treatment efficiency elevation.…”

Recent advances in fluorescent probes for lipid droplets

“…5). 21 The probe displayed a large solvatochromic shift from 498 nm (in hexane) to 625 nm (in acetonitrile), showing significant ICT character. However, 9 exhibited a relatively high quantum yield (40.8) in both hexane and acetonitrile; even in more polar solvents, its quantum yield was 40.56.…”

“…Especially, the polarity of LDs was reported to be close to n-hexane, toluene or dioxane via the spectral images. 21,29,30,39,48,54,57,59 Thus, the dielectric constant of LDs was estimated to be about 1-3. (5) LD probes for special microscopies, such as TP, FLIM and SETD, provided higher imaging quality.…”

A New Lipid‐Droplets‐Targeted Fluorescence Probe with Dual‐Reactive Sites for Specific Detection of ClO− in Living Cells

“…[17,18] The traditional imaging techniques, such as Raman microscopy, transmission electron microscopy, and immunofluorescence microscopy, [19][20][21] have been employed for the purpose to visualize lipid droplets, but some challenges have still been existed, such as complicated procedures and poor cellular permeability and thus may disturb the functions of cells. [22][23][24] Keeping these disadvantages in mind, in recent years, fluorescence imaging has been disclosed as a reliable tool for visualizing the localization and the dynamics of cellular compartments. [25][26][27] Especially, by utilizing the aggregationinduced emission (AIE) active dyes (named as AIEgens), the cell imaging could be accomplished with excellent selectivity, remarkable sensitivity, and extraordinary temporal spatial resolution.…”