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Abstract：
Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the power- and gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.
摘要：
背景：脂滴（LD）是与真核细胞中许多生理过程相关的关键细胞器。为了可视化和研究LD，荧光成像技术，包括共焦成像以及新兴的受激发射损耗超分辨率成像（STED），被认为是最有用的方法。然而,直接受限于先进的LD荧光探针的可用性，由于LD的快速研究进展，LD荧光成像的性能越来越不令人满意。方法：我们在此新开发了一种名为Lipi-QA的高级LDs荧光探针，作为LDs荧光成像和生物学研究的有力工具。在四种细胞系中进行Lipi-QA和LDs荧光探针Ph-Red的共定位成像。还通过与市售LD探针尼罗红比较来评估Lipi-QA的LD染色选择性和光稳定性。通过时间门控检测确定LD中Lipi-QA的原位荧光寿命。通过MTT分析评估Lipi-QA的细胞毒性。通过LeicaSP8STED超分辨率纳米显微镜测试了STED饱和强度以及功率和门时间依赖性分辨率。然后设计了延时3D共焦成像和延时STED超分辨率成像来研究LD的复杂生理功能。结果：具有超光稳定性的优点，高LD选择性，长荧光寿命和低STED饱和强度，荧光探针Lipi-QA能够进行长期延时三维(3D)共聚焦成像以原位监测3D空间中的LD和延时STED超分辨率成像(高达500个STED帧)以跟踪纳米级分辨率(37nm)的LD动态.结论：基于最先进的荧光成像结果，已成功提供了一些有关LD的新生物学见解。例如,长期延时3D共聚焦成像无疑回答了一个重要且有争议的问题，即在饥饿刺激下，LD的数量将显着减少而不是增加；具有最高分辨率的延时STED超分辨率成像首次令人印象深刻地揭示了纳米级LD的裂变过程；STED超分辨率成像在纳米级进一步揭示了饥饿引起的LD尺寸和速度变化。所有这些结果不仅突出了新开发的荧光探针的实用性，而且显着促进了LD的生物学研究。
