中国激光, 2023, 50 (9): 0907102, 网络出版: 2023-04-24  

具有荧光开关特性的喹啉衍生物用于高效选择性标记细胞脂滴 下载: 514次

Quinoline Derivatives with Fluorescent Switching Properties for Efficient Selective Labeling of Cellular Lipid Droplets
作者单位
吉林大学电子科学与工程学院集成光电子学国家重点实验室,吉林 长春 130012
摘要
脂滴是一种重要的细胞器,与细胞中多种生理活动密切相关。为了观察脂滴并研究其多种多样的功能,共聚焦荧光成像技术是最有力的工具之一。然而,细胞脂滴荧光成像所需的具有高荧光亮度和高标记选择性的脂滴荧光探针却十分有限,这严重限制了脂滴的深入研究。研制了一种具有荧光开关特性的喹啉衍生物脂滴荧光探针(Lipi-QL)。该探针因其敏感的极性淬灭荧光特性,展现出了很高的脂滴标记选择性。Lipi-QL能靶向脂滴是因为其脂溶性,进入脂滴后,荧光增强是脂滴内的较低极性引起的。给受体型分子结构还赋予了该探针高的荧光亮度以及大的斯托克斯位移。将该探针用于细胞脂滴共聚焦荧光成像,在不同浓度下都实现了显著优于脂滴商用探针BODIPY 493/503的标记选择性。此外,使用该荧光探针实现了固定细胞的三维共聚焦成像和活细胞的四色共聚焦成像。该荧光探针的研制一方面为脂滴生理功能的研究提供了强有力的工具,另一方面也为新型高标记选择性荧光探针的设计提供了新的思路。
Abstract
Objective

Lipid droplets are important organelles closely associated with various cellular physiological activities. Confocal fluorescence imaging is a powerful tool for observing lipid droplets and studying their diverse functions. However, lipid droplet fluorescent probes with the high fluorescence intensity and labeling selectivity required for cellular lipid droplet fluorescence imaging are limited, severely limiting the in-depth study of lipid droplets. In this study, we develop Lipi-QL, a quinoline-derivative lipid droplet fluorescent probe with fluorescence-switching properties.

Methods

The probe exhibits high selectivity for lipid droplet labeling owing to its sensitive polar quenching fluorescence properties. The donor-type molecular structure also confers high fluorescence intensity and large Stokes shifts on the probe. When using this probe for confocal fluorescence imaging of cellular lipid droplets, significantly better labeling selectivity is achieved at varying concentrations than when using the commercial BODIPY 493/503 lipid droplet probe. Additionally, three-dimensional confocal imaging of fixed cells and four-color confocal imaging of live cells are performed using this fluorescent probe. The development of this probe provides a powerful tool for studying the physiological functions of lipid droplets and provides a new idea for the design of new highly labeled selective fluorescent probes.

Results and Discussions

As shown in Fig.1(c), the probe exhibits highly efficient fluorescence emission when the water volume fraction is 0, indicating that it can exhibit high fluorescence intensity within lipid droplets. When the water volume fraction gradually increases, the probe exhibits extremely sensitive fluorescence quenching properties: quenching most of the fluorescence emission when the water volume fraction is only 1%. When the water volume fraction increases to 20%, the probe's emission is almost completely quenched, and the fluorescence signal disappears. This indicates that even if a small portion of the probe enters the cell and stains organelles other than lipid droplets, the fluorescence emission is quenched by the polar environment in which it is placed, thus showing a high selectivity for lipid droplet staining. We also test the fluorescence switching characteristics of the commercial lipid droplet dye, BODIPY 493/503. As shown in Fig.1(d), the fluorescence quenching of BODIPY 493/503 in the dioxane solution with 40% water volume fraction is not apparent, which may be the main reason for its poor lipid droplet staining selectivity. Figure 3 shows that the Lipi-QL fluorescent probe efficiently stains cellular lipid droplets at different concentrations. In contrast, BODIPY 493/503 stains lipid droplets much less selectively, staining other membrane-like cellular structures in addition to cellular lipid droplets with a lower imaging signal-to-noise ratio. This staining selectivity comparison highlights the significant advantage of the polar quenching luminescence property of the Lipi-QL fluorescent probe for the efficient and selective labeling of cellular lipid droplets. After washing the free probe with phosphate buffered saline (PBS), three-dimensional confocal imaging is performed. The experiment is performed at a high xy-plane point resolution with a small z-sweep step (200 nm) to obtain high-quality 3D confocal photographs (Fig.4). The spatial distribution of intracellular lipid droplets can be seen clearly in this photograph, demonstrating the usefulness of the probe for 3D confocal imaging. The Lipi-QL fluorescent probe is also used for multicolor confocal imaging because of its excellent performance. The nuclei, lipid droplets, lysosomes, and mitochondria of live HeLa cells are stained with the Hoechst 33342 commercial dye for nuclei, Lipi-QL commercial dye for lipid droplets, LysoTracker Deep Red commercial dye for lysosomes, and MitoTracker Deep Red commercial dye for mitochondria, respectively. High-quality four-color confocal images of living cells are successfully obtained by performing confocal fluorescence. Based on the different absorption and emission spectra of these four fluorescent probes, imaging is performed through line-by-line scanning, effectively avoiding the occurrence of crosstalk between individual fluorescent channels.

Conclusions

In conclusion, an advanced lipid droplet fluorescent probe with fluorescence switching properties, Lipi-QL, is developed in this study, which allows for the efficient and selective labeling of cellular lipid droplets. The probe also has high fluorescence brightness, a large Stokes shift, and good biocompatibility. Based on these excellent properties, high-quality three-dimensional confocal imaging of fixed cells and four-color confocal imaging of live cells are successfully achieved using this probe, highlighting its utility in lipid droplet fluorescence imaging. The development of this probe provides an effective tool for cell biology studies of lipid droplets and a new approach for the design and synthesis of highly labeled selective fluorescent probes.

赵力, 周日, 刘冠男, 彭桂衫, 王晨光, 贾晓腾, 卢革宇. 具有荧光开关特性的喹啉衍生物用于高效选择性标记细胞脂滴[J]. 中国激光, 2023, 50(9): 0907102. Li Zhao, Ri Zhou, Guannan Liu, Guishan Peng, Chenguang Wang, Xiaoteng Jia, Geyu Lu. Quinoline Derivatives with Fluorescent Switching Properties for Efficient Selective Labeling of Cellular Lipid Droplets[J]. Chinese Journal of Lasers, 2023, 50(9): 0907102.

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