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周日; 王晨光; 卢革宇

doi:10.37188/CO.2022-0077

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doi: 10.37188/CO.2022-0077

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

基金项目:国家自然科学基金（No. 61831011，No. 62075079）

详细信息

作者简介:
周　日（1992—），男，吉林德惠人，博士，2021年于吉林大学获得博士学位，主要从事细胞脂滴荧光探针的研制及其STED超分辨成像研究。E-mail： [email protected]

王晨光（1988—），男，安徽利辛人，教授，博士生导师，2013年于吉林大学获得博士学位，主要从事有机荧光探针的研制及其STED超分辨成像研究。E-mail：[email protected]

卢革宇（1963—），男，黑龙江海伦人，教授，博士生导师，1998年于日本九州大学获得博士学位，主要从事传感器、光电子材料与器件、物联网和微纳传感系统等方面的研究。E-mail：[email protected]

中图分类号:O652.7;O439
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- 收稿日期: 2022-04-22
- 修回日期: 2022-05-07
- 网络出版日期: 2022-06-20

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State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and Engineering, Jilin University, Changchun 130012, China

Funds:Supported by National Natural Science Foundation of China (No. 61831011, No. 62075079)

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Corresponding author: [email protected]; [email protected]

摘要

摘要:
脂滴是真核细胞中必不可少的一种球形细胞器，与很多细胞生理学过程息息相关。荧光成像技术是观察研究脂滴最有力的工具之一。受光学衍射极限的限制，传统的宽场以及共聚焦显微镜所能达到的成像分辨率约为250 nm左右，这对于观测小脂滴，尤其是新生脂滴（尺寸约30~60 nm）来说是远远不够的。在这种情况下，近年来新兴的各种能够打破衍射极限的超分辨荧光显微镜（如受激发射损耗显微镜、结构光照明显微镜以及光激活定位显微镜等）逐渐吸引了科研人员的兴趣。为了得到高分辨率脂滴荧光图像，除了上述超分辨显微镜之外，还需要具有与之相匹配的高性能荧光探针。本文将简要介绍这几种超分辨显微镜的工作原理，讨论其对荧光探针光物理性质的特殊要求，并进一步系统总结脂滴超分辨成像荧光探针的研究进展。与此同时，本文将分析对比不同超分辨显微镜在脂滴荧光成像方面的优势与不足，并对其发展趋势进行展望。
- 脂滴 /
- 超分辨成像 /
- 受激发射损耗显微镜 /
- 结构光照明显微镜 /
- 光激活定位显微镜 /
- 荧光探针
Abstract:
Lipid droplets are a kind of spherical organelle in eukaryotic cells and are relevant to many cellular physiological processes. Fluorescence imaging techniques are one of the most powerful tools to visualize and study lipid droplets. However, conventional wide-field microscopy and confocal microscopy can only provide a resolution of about 250 nm due to the limitation of optical diffraction. This resolution is quite insufficient for visualizing the small lipid droplets, especially the nascent ones (size of about 30~60 nm). Emerging super-resolution microscopes that can break the diffraction limit (such as stimulated emission depletion microscopy, structured illumination microscopy and photoactivated localization microscopy) have gradually attracted much interest in recent years. To obtain high-resolution fluorescence images of lipid droplets, the advanced fluorescent probes which meet the special requirements of the corresponding super-resolution microscopes are highly essential. This review paper will briefly introduce the working principles of various super-resolution microscopes, discuss the special requirements on the photophysical properties of fluorescent probes, and systematically summarize the research progress of super-resolution imaging of lipid droplets by employing these fluorescent probes. Meanwhile, this review will compare the advantages and shortcomings of different super-resolution techniques for lipid droplets imaging, and prospect their future possible trends.
- lipid droplets /
- super-resolution imaging /
- stimulated emission depletion microscopy /
- structured illumination microscopy /
- photoactivated localization microscopy /
- fluorescent probes

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图 1 STED超分辨显微镜原理

Figure 1. The principle of the STED super-resolution microscope

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图 2 用于脂滴STED超分辨成像的有机荧光探针

Figure 2. The organic fluorescent probes for STED super-resolution imaging of lipid droplets

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图 3 脂滴荧光探针LAQ1在多色共聚焦成像和STED超分辨成像中的应用。（a）脂滴、内质网和线粒体的三色荧光成像；（b）穿过白色箭头的信号强度分布；（c）小脂滴的STED成像；（d）穿过同一小脂滴的共聚焦和STED成像信号强度图^{[
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Figure 3. The applications of lipid droplets fluorescent probe LAQ1 in multicolor confocal imaging and STED super-resolution imaging. (a) Three-color fluorescence imaging of lipid droplets, endoplasmic reticulum and mitochondria; (b) signal intensity profiles across the white arrow; (c) STED imaging of lipid droplets; (d) confocal and STED imaging signal intensity profiles across the same lipid droplet^{[
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图 4 脂滴荧光探针DTPA-BT-M在（a）STED超分辨和（b）双光子成像中的应用^{[
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Figure 4. The applications of lipid droplets fluorescent probe DTPA-BT-M in (a) STED super-resolution and (b) two-photon imaging^{[
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图 5 脂滴荧光探针Lipi-DSB的STED超分辨成像。（a）损耗激光功率依赖的分辨率；（b）与线粒体荧光探针四甲基罗丹明搭配的双色STED成像；（c）连续1000帧的STED超分辨成像动态跟踪；（d）STED超分辨成像动态跟踪纳米尺度新生脂滴的融合过程^{[
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Figure 5. STED super-resolution imaging of lipid droplets fluorescent probe Lipi-DSB. (a) STED laser power-dependent resolution; (b) two-color STED imaging of Lipi-DSB paired with mitochondria fluorescent probe tetramethylrhodamine; (c) time-lapse STED super-resolution imaging (1000 consecutive frames); (d) dynamic tracking of nascent lipid droplet fusion processes at the nanoscale by STED super-resolution imaging^{[
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图 6 脂滴荧光探针Lipi-BDTO的共聚焦和STED超分辨成像。（a）共聚焦和STED成像对比；（b）3D STED成像^{[

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Figure 6. Confocal and STED super-resolution imaging of the lipid droplets fluorescent probe Lipi-BDTO. (a) Comparison of confocal and STED imaging; (b) 3D STED imaging^{[
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图 7 SIM超分辨成像原理^{[
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Figure 7. The principle of SIM super-resolution microscope^{[

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图 8 用于脂滴SIM超分辨成像的有机荧光探针

Figure 8. The organic fluorescent probes for SIM super-resolution imaging of lipid droplets

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图 9 脂滴荧光探针LD-FG的SIM超分辨成像。（a）缓冲策略提升光稳定性；（b）荧光探针的光开关特性；（c）相邻脂滴间的融合；（d）不相邻脂滴间的融合^{[
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Figure 9. SIM super-resolution imaging of the lipid droplets fluorescent probe LD-FG. (a) Buffer strategy enables stable imaging of lipid droplets; (b) fluorescence switching property of the fluorescent probe; (c) coalescence between adjacent lipid droplets; (d) coalescence between non-adjacent lipid droplets^{[
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图 10 有机荧光探针在脂滴SIM超分辨成像中的应用。（a）荧光探针DTZ-TPA-DCN用于染色核脂滴^{[
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]}；（b）荧光探针NIM-3A用于分析细胞内脂滴数量和尺寸^{[
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Figure 10. Applications of organic fluorescent probes in SIM super-resolution imaging of lipid droplets. (a) Fluorescent probe DTZ-TPA-DCN used for staining nuclear lipid droplets^{[
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]}; (b) fluorescent probe NIM-3A used for analyzing the number and size of lipid droplets^{[
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]}; (c) fluorescent probe NIM-7 used for tracking dynamic processes of lipid droplets and lysosomes^{[
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图 11 分子定位技术原理示意图^{[
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Figure 11. Schematic diagram of molecular localization technology^{[
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图 12 光开关荧光探针在脂滴PALM超分辨成像中的应用。（a）具有荧光开关特性的有机荧光探针；（b）脂肪细胞中脂滴在宽场和PALM显微镜下的明场和双色成像^{[
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Figure 12. Applications of light-switchable fluorescent probes in PALM super-resolution imaging of lipid droplets. (a) The organic fluorescent probes with fluorescence ON/OFF characteristics; (b) bright field and two-color imaging of lipid droplets in adipocyte under wide-field and PALM microscopes^{[
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表 1 文献中报道的细胞脂滴超分辨成像结果总结

Table 1. Summary of super-resolution imaging of cell lipid droplets reported in the literatures

发表年份	超分辨成像技术	荧光探针	成像分辨率/nm	是否动态超分辨成像	参考文献
2021	STED	LAQ1	166	未提及	19
2021	STED	DTPA-BT-M	95	未提及	20
2021	STED	Lipi-DSB	58	是	21
2021	STED	Lipi-BDTO	65	是	22
2021	SIM	LD-FG	180	是	27
2021	SIM	DTZ-TPA-DCN	未提及	未提及	28
2018	SIM	NIM-3A	未提及	未提及	29
2019	SIM	NIM-7	未提及	未提及	30
2019	PALM	SNile Red	<250	未提及	31
2021	PALM	BAD-Oxm	未提及	未提及	32
2020	PALM	BODIPY-C12	125	未提及	33

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