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超分辨显微成像技术按照其成像特点可分为三类:一是基于压缩点扩散函数的STED显微成像技术 [16];二是SIM技术[17];三是基于荧光分子开关效应的SMLM技术[18],以光激活定位显微成像(Photo activation localization microscopy, PALM)和随机光学重建显微成像(Stochastic optical reconstruction microscopy, STORM)技术为代表。根据庄小威等[19]的综述总结以上超分辨显微成像技术的特点,见表1。
表 1 三类超分辨显微成像技术的原理及特点
Table 1. Principles and characteristics of three types of super-resolution microscopy
Principle xy resolution/nm z resolution/nm Fluorescent
moleculesLive cell imaging Image reconstruction STED Stimulated emission depletion based on PSF ~40 ~70 Free Yes Not required SIM Structured illumination ~50 ~250 Free Yes Required PALM Photo activated localization based on
photoswitchable fluorescent molecules~20 ~50 Photoswitchable Yes Required STORM Stochastic optical reconstruction based on
photoswitchable fluorescent molecules~20 ~50 Photoswitchable Yes Required -
STED显微成像技术以激光共聚焦技术为基础,它通过激发态荧光团的损耗来提高成像分辨率。分辨率取决于激发点的大小,即点扩散函数(Point spread function, PSF),由于PSF内的所有荧光团都会对测量信号产生影响,STED通过引入另一束环形损耗光包围激发光,抑制PSF外围荧光团的信号发射,有效减小激发光斑尺寸,从而提高成像分辨率[16, 20-21](图1)。STED利用纯光学的方法实现超分辨成像,因此它不需要对图像进行后续处理。同时,其分辨率相对较高,可达到40 nm左右的横向分辨率以及70 nm左右的纵向分辨率,而且它的成像速度相对较快,对荧光染料的特异性要求不高[22]。基于以上特点,STED已成为细胞及细胞器精细结构研究中的理想选择之一。但是,STED采用逐点扫描的采集方式导致其时间分辨率仍不能满足活跃的细胞活动,如高度动态的细胞器相互作用过程的成像,这也就限制了其在细胞器相互作用研究中的应用。
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SIM采用结构化的条纹图案进行照明,通过莫尔效应使传统荧光显微镜无法探测到的样品高频信息进入成像系统的频率探测范围内,实现超分辨成像[23-26](图2)。最初,SIM基于宽场结构光照明,受线性荧光激发特性的影响,其分辨率较宽场成像只能提高两倍,并不能无限制提高。随着非线性荧光激发技术的发展,SIM的横向分辨率获得显著提升,可达到50 nm。虽然宽场结构光照明成像速度快,视场大,但其在厚组织样本中应用受限。近来的研究中,点扫描结构光照明技术使SIM的成像深度有所提升,同时得益于硬件设备的升级和去卷积算法的优化,SIM在保持分辨率的前提下有效提升了成像速度。点扫描SIM还可以与双光子激发相结合,实现厚组织的高分辨率观察[27]。由于SIM具有良好的荧光探针适用性、较高的成像分辨率以及较快的成像速度,已成为活细胞观察中使用最广泛的超分辨成像方法[28-29]。
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SMLM使用特定的荧光探针,特定波长的激光来激活荧光分子,然后用另一波长激发荧光成像,通过控制激光强度使少量荧光分子激活,采集大量图像后利用单分子定位算法定位单分子的中心位置,并叠加重建得到超分辨图像[18, 30-31](图3),其典型的代表是PALM,可达到10~80 nm的横向分辨率。此外,基于单分子定位技术,庄小威等[32]提出了STORM,通过荧光分子的on-off开关状态采集信号并获得超分辨图像,可实现20~50 nm的横向分辨率。单分子定位显微成像技术已广泛用于组织和细胞成像研究,在细胞器精细结构、蛋白分子的运动等观察中优势明显,它可以呈现微管、线粒体、脂滴甚至核孔复合体等的超微结构,具有观察装置简单、操作方便、分辨率高等优势,但其成像速度低,在活细胞成像中不具优势,且对荧光分子特异性要求较高[33]。
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超分辨显微成像技术突破了光学衍射极限,使活细胞及其细胞器成像分辨率有了显著的提升,为从细胞器层面研究细胞功能提供了新手段。通过总结现有的部分研究发现(表2),结构光照明显微成像(SIM)在细胞器相互作用研究中更受青睐,其次是受激发射损耗显微成像(STED),而单分子定位显微成像(SMLM)在活细胞中细胞器相互作用研究中应用少见,可能与其时间分辨率不足有关。
表 2 超分辨显微成像技术在细胞器相互作用研究中的应用
Table 2. Application of super-resolution microscopy in the study of organelle interactions
Organelle interactions Research contents Super-resolution microscopy Cells Mitochondria-lysosome Mitochondria and lysosome contact ( MLC) marks sites of mitochondrial fission and the formation and release of MLC is bidirectionally regulated by mitochondrial and lysosomal dynamic [36]; Endoplasmic reticulum (ER) recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria [37]; The analysis methods of mitochondria-lysosome interactions based on structured illumination microscopy
(SIM) [38]; Lysosome-targeted biosensor for the super-resolution imaging of lysosome-mitochondrion interaction based on SIM[39].SIM HT22 cells; SHSY-5Y cells; HeLa cells; HSF cells Mitochondria- nucleus Observation of mitochondria-nucleus contact and the change of expression sites of Sirt4 from mitochondria to nucleus under mitochondrial stress conditions [43-44]. SIM HeLa cells; Pancreatic β cells Mitochondria-cytoskeleton Mitochondrial morphology and distribution are regulated by cytoskeleton. During mitosis, dense meshwork of subcortical actin cables organizes three-dimensional mitochondrial positioning to ensure both equal and random inheritance of mitochondria in symmetrically dividing cells [48]; Actin maintains microtubule organization, dynamics and stability by affecting tubulin acetylation levels and further regulate mitochondrial distribution [49]; Cytoskeleton regulates fission and fusion of mitochondria [50]. SIM; STED Hela cells; COS-7 cells; HEK293 cells; U2 OS cells Mitochondria-lipid droplet Observation of mitochondria-lipid droplets contacts; Overexpression of perilipin5 leads to increased number of mitochondria surrounding lipid droplets [55]. STED COS-7 cells Mitochondria-Endoplasmic reticulum Tubular endoplasmic reticulum regulates mitochondrial fission and fusion [64]; Calcium transients on the sites of mitochondria-endoplasmic reticulum contacts[67]; In SEPN1-related myopathy, SEPN1 deficiency results in less mitochondria-endoplasmic reticulum contacts, calcium contents and damaged oxidative phosphorylation process [68]. SIM COS-7 cells; U2 OS cells; HeLa cells Endoplasmic reticulum-lysosome Lysosomes moved synchronously with local endoplasmic reticulum. The anchorage of lysosomes to endoplasmic reticulum growth tips is critical for endoplasmic reticulum tubule elongation and connection[15]; Endoplasmic reticulum contacts with the edge of lysosome, which promotes the long-distance transportation of lysosome[64]. SIM COS-7 cells; U2 OS cells Endoplasmic reticulum-cytoskeleton Endoplasmic reticulum anchors to microtubules, which guides the formation of new endoplasmic reticulum tubule branches[64]; Endoplasmic reticulum dynamics play important roles in microtubules distribution[15]. SIM; STED COS-7 cells; U2 OS cells 超分辨显微成像技术在细胞器及其相互作用研究中的贡献主要体现在以下三方面:获得细胞器结构的直观精细高分辨率图像;为已发现的细胞器相互作用提供了高分辨率图像直接证据;观察到细胞器间相互作用的新形式。超分辨显微成像技术虽然在细胞器精细结构观察与细胞器相互作用研究中具有其他技术不能替代的作用,但由于成像的分辨率、速度和成像深度,以及荧光探针的光毒性等问题(表3),在实际应用中仍存在一些不足,限制了其在该领域内的应用效果。
表 3 超分辨显微成像技术的应用参数
Table 3. Application parameters of super-resolution microscopy
STED SIM SMLM xy resolution/nm ~40 ~50 ~20 z resolution/nm ~70 ~250 ~50 Temporal resolution 5 ms-2 s 10-500 ms 1 min-1 h Light intensity Medium-high Low-medium Low-medium Live cell dynamic imaging Medium Good Poor (1) STED在对活细胞细胞器相互作用研究中,在横向分辨率上突破了衍射极限,但由于该技术采用的是点扫描技术,在对大视野区域进行成像时,图像采集比普通荧光显微镜耗时长,限制了面积较大的活细胞等生物样本内生物分子的动态过程或相互作用的实时成像;同时由于使用高功率的损耗光,产生的光毒性会对生物活性样本产生一定的光损伤,限制了细胞器及相互作用成像的准确性,因此可从提高成像速度和降低损耗光功率两方面改进,进一步提高STED在活细胞中细胞器及相互作用研究中的适用性。
(2) SIM在对活细胞细胞器相互作用研究中,在横向与纵向分辨率上均有所提高,可实现活细胞中细胞器的3D高分辨率成像,同时具有高速扫描、多色成像以及低光漂白和低光毒性等优势,非常适合研究细胞器动力学。与TIRF或TIRF-SIM相比,GI-SIM荧光强度更大;与SDCM相比,它提供了更好的空间分辨率和更快的成像速度;与其他超分辨显微成像技术相比,它使时空分辨率与光漂白和光毒性之间更好地平衡。然而由于只能将分辨率提高到宽场成像两倍,因此提高SIM的成像分辨率可提高其在细胞器相互作用中的应用。
(3)目前单分子定位显微成像SMLM,主要包括光激活定位显微成像(PALM)与随机光学重构显微成像(STORM)。该技术可将分辨率提高到20 nm以下,PALM技术通过细胞自身表达荧光蛋白进行成像,更适合用于活细胞内蛋白的超分辨成像。SMLM在荧光团本身稀疏标记的样本中,可用于活样本中进行超分辨率跟踪,还可实现活体样本的3D成像。但是由于分子定位具有顺序性,需要反复激活-淬灭荧光分子获得许多幅原始图像重构获得超分辨图像,因此其成像的时间分辨率相对较低,限制了其在较为活跃的细胞器相互作用成像中的应用。
超分辨显微成像技术的应用为活细胞中细胞器相互作用研究提供了新手段,然而现有研究中仍存在一些不足,如多细胞相互作用成像受到空间分辨率的限制,动态变化的成像受到时间分辨率的限制,高功率激发光产生的光毒性无法避免对活细胞产生影响等。因此,未来超分辨显微成像技术在活细胞的细胞器相互作用研究应用中,将在提高空间和时间分辨率,降低激发光功率,开发低光毒性荧光染料,降低光毒性对活细胞的影响等方面进一步提升。此外,人工智能算法在超分辨显微成像技术中的应用愈加广泛,如何利人工智能算法来提升成像的效果以及分析细胞器相互作用规律也将是重点研究的方向之一。总之,超分辨显微成像技术的出现为揭示细胞器相互作用规律提供了有力的技术手段,对于探索细胞器互作异常引发疾病背后的机制具有非常重要的意义。
Application of super-resolution microscopy in the study of organelle interactions (invited)
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摘要: 观察细胞器间动态相互作用,深入分析作用规律,对于揭示生理病理过程现象背后的机制具有十分重要的意义。传统光学显微镜受到由光波波长和孔径造成的衍射极限的限制,无法观测细胞器纳米级精细结构及细胞器间相互作用的动态变化规律。超分辨显微成像技术的出现为细胞器相互作用研究提供了重要手段,在深入揭示细胞器相互作用规律,阐明生理病理现象深层的机制研究中发挥了重要的作用。文中介绍了受激发射损耗(Stimulated emission depletion, STED)显微成像、结构光照明显微成像(Structured illumination microscopy, SIM)、单分子定位显微成像(Single molecule localization microscopy, SMLM)技术,并总结了这三类超分辨显微成像技术在细胞器相互作用中的应用与现状,为超分辨显微成像技术在细胞器相互作用研究中的应用提供思路拓展。最后,对超分辨显微成像技术在细胞器相互作用研究中的优势与不足进行分析总结,展望了超分辨显微成像技术在活细胞内细胞器相互作用成像中的需求发展趋势,为光学与医学及生物学的交叉融合发展提供一定的参考。
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关键词:
- 超分辨显微成像 /
- 细胞器间相互作用 /
- 受激发射损耗显微成像 /
- 结构光照明显微成像 /
- 单分子定位显微成像
Abstract: Observing dynamic interaction between organelles and analyzing the law of action is of great significance for revealing the mechanism behind the phenomenon of physiological and pathological processes. Due to the limitation of the optical diffraction determined by wavelength and aperture, traditional optical microscopes cannot observe the nanoscale fine structure of organelles and the dynamic changes of interactions among them. The emergence of super-resolution microscopy imaging technology provides an important mean for the study of organelle interaction. This paper introduces the fluorescence microscopy (STED), structured illumination imaging (SIM), and single-molecule localization imaging (SMLM). The application of these super-resolution microscopy in the study of dynamic interaction between organelles provides the expansion of application ideas for super-resolution microscopy. Finally, the advantages and disadvantages of super-resolution microscopy in the study of organelle interactions are analyzed. In conclusion, the demand and development trend of super-resolution microscopy technology in the imaging of intracellular organelle interaction in living cells is prospected, which provides a certain reference for the cross-integration development of optics, medicine and biology. -
图 6 STED观察线粒体-脂滴接触。(a) 接触面PLIN5的表达;(b) 线粒体-脂滴接触区渲染图(绿色:脂滴;红色:线粒体;蓝色:接触区)[55-56]
Figure 6. STED showed the mitochondrion-lipid droplet interface. (a) Expression of PLIN5 (red) at interface of mitochondria and lipid droplet; (b) Voxelized rendering of extracted LD, mitochondria and overlapping zones between them (LD (green), mitochondria (red) and computed contact zones (blue))[55-56]
表 1 三类超分辨显微成像技术的原理及特点
Table 1. Principles and characteristics of three types of super-resolution microscopy
Principle xy resolution/nm z resolution/nm Fluorescent
moleculesLive cell imaging Image reconstruction STED Stimulated emission depletion based on PSF ~40 ~70 Free Yes Not required SIM Structured illumination ~50 ~250 Free Yes Required PALM Photo activated localization based on
photoswitchable fluorescent molecules~20 ~50 Photoswitchable Yes Required STORM Stochastic optical reconstruction based on
photoswitchable fluorescent molecules~20 ~50 Photoswitchable Yes Required 表 2 超分辨显微成像技术在细胞器相互作用研究中的应用
Table 2. Application of super-resolution microscopy in the study of organelle interactions
Organelle interactions Research contents Super-resolution microscopy Cells Mitochondria-lysosome Mitochondria and lysosome contact ( MLC) marks sites of mitochondrial fission and the formation and release of MLC is bidirectionally regulated by mitochondrial and lysosomal dynamic [36]; Endoplasmic reticulum (ER) recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria [37]; The analysis methods of mitochondria-lysosome interactions based on structured illumination microscopy
(SIM) [38]; Lysosome-targeted biosensor for the super-resolution imaging of lysosome-mitochondrion interaction based on SIM[39].SIM HT22 cells; SHSY-5Y cells; HeLa cells; HSF cells Mitochondria- nucleus Observation of mitochondria-nucleus contact and the change of expression sites of Sirt4 from mitochondria to nucleus under mitochondrial stress conditions [43-44]. SIM HeLa cells; Pancreatic β cells Mitochondria-cytoskeleton Mitochondrial morphology and distribution are regulated by cytoskeleton. During mitosis, dense meshwork of subcortical actin cables organizes three-dimensional mitochondrial positioning to ensure both equal and random inheritance of mitochondria in symmetrically dividing cells [48]; Actin maintains microtubule organization, dynamics and stability by affecting tubulin acetylation levels and further regulate mitochondrial distribution [49]; Cytoskeleton regulates fission and fusion of mitochondria [50]. SIM; STED Hela cells; COS-7 cells; HEK293 cells; U2 OS cells Mitochondria-lipid droplet Observation of mitochondria-lipid droplets contacts; Overexpression of perilipin5 leads to increased number of mitochondria surrounding lipid droplets [55]. STED COS-7 cells Mitochondria-Endoplasmic reticulum Tubular endoplasmic reticulum regulates mitochondrial fission and fusion [64]; Calcium transients on the sites of mitochondria-endoplasmic reticulum contacts[67]; In SEPN1-related myopathy, SEPN1 deficiency results in less mitochondria-endoplasmic reticulum contacts, calcium contents and damaged oxidative phosphorylation process [68]. SIM COS-7 cells; U2 OS cells; HeLa cells Endoplasmic reticulum-lysosome Lysosomes moved synchronously with local endoplasmic reticulum. The anchorage of lysosomes to endoplasmic reticulum growth tips is critical for endoplasmic reticulum tubule elongation and connection[15]; Endoplasmic reticulum contacts with the edge of lysosome, which promotes the long-distance transportation of lysosome[64]. SIM COS-7 cells; U2 OS cells Endoplasmic reticulum-cytoskeleton Endoplasmic reticulum anchors to microtubules, which guides the formation of new endoplasmic reticulum tubule branches[64]; Endoplasmic reticulum dynamics play important roles in microtubules distribution[15]. SIM; STED COS-7 cells; U2 OS cells 表 3 超分辨显微成像技术的应用参数
Table 3. Application parameters of super-resolution microscopy
STED SIM SMLM xy resolution/nm ~40 ~50 ~20 z resolution/nm ~70 ~250 ~50 Temporal resolution 5 ms-2 s 10-500 ms 1 min-1 h Light intensity Medium-high Low-medium Low-medium Live cell dynamic imaging Medium Good Poor -
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