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摘要:为了进一步认知复杂环境中的细胞生物学过程，研究人员发展了各种各样的生物成像技术。在这些技术中，生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而，传统的荧光成像技术受到了光学衍射极限的限制，无法分辨低于200 nm的空间结构，阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制，能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外，目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。Abstract:In order to further understand the biological cellular processes in the complex environments, a variety of bioimaging techniques have been developed by researchers. Biofluorescence imaging has been extensively developed due to its simple imaging conditions and compatibility with biological samples. However, the traditional fluorescence imaging technology is restricted by the optical diffraction limit, so it is impossible to resolve the spatial structure below 200 nm, which hinders the study of the biological processes of subcellular structures. Super-resolution fluorescence microscopy breaks through the limitations of imaging resolution with traditional optical diffraction and can acquire nanoscale cellular dynamics. In addition to improvements and upgrades to traditional wide-field fluorescence microscope frames, typical super-resolution imaging microscopy techniques currently also rely on the photophysical properties of fluorescent probe materials. Commonly used fluorescent probe materials mainly include fluorescent proteins, organic fluorescent molecules and fluorescent nanomaterials. This paper introduces several mainstream super-resolution fluorescence microscopy techniques and summarizes the application status of fluorescent probe materials that have been successfully applied to super-resolution biofluorescence imaging.
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图 1不同生物成像技术空间分辨率对比图
Figure 1.Spatial resolution comparison of different biological imaging techniques
图 3红光发射的罗丹明荧光分子结构式
Figure 3.Chemical structures of red-emitting rhodamine fluorophores
图 4用于STED荧光显微镜的荧光分子1及其衍生物结构式
Figure 4.Chemical structures of fluorescent molecular 1 and derivatives used in STED microscopy
图 5荧光分子2和3的化学结构式及在活细胞中的正交反应示意图
Figure 5.Chemical structures of fluorescent moleculars 2 and 3 and the bioorthogonal reaction in live cells
图 7光激活定位显微镜和随机光学重构显微镜工作原理及亚细胞结构超分辨图(左侧：PALM及COS-7细胞溶酶体超分辨图像；右侧：STORM原理图及亚细胞结构单色、多色超分辨图像)[14,17]
Figure 7.Working principle of Photoactivation Localization Microscopy, Stochastic Optical Reconstruction Microscopy and subcellular structure super resolution images(Left: PALM and COS-7 cell lysosomal superresolution images; right:STORM schematic and subcellular structure monochrome, multicolor super-resolution images)[14,17]
图 9罗丹明衍生物(11~21)光诱导转换过程分子结构式
Figure 9.Photoinduced reversible switching process of rhodamine derivatives(11~21)
图 10Carbofluoresceins，Carborhodamines110的分子结构式
Figure 10.Chemical structures of carbofluoresceins, carborhodamines110(28)
图 12(a~c)3种不同DNA结构的三色STORM图像(Alexa405/Cy5, Cy2/Cy5和Cy3/Cy5标记；405 nm, 457 nm和532 nm 激发)；(d)Alexa647/Cy3标记的BC-S-1细胞内网格蛋白凹坑(CCPs)的三维STORM图像[54-55]
Figure 12.(a~c)Three-color STORM images of three different DNA constructs(labelled with Alexa405/Cy5, Cy2/Cy5, and Cy3/Cy5, activated using 405, 457 and 532 nm laser, respectively); (d)3D STORM image of clathrin-coated pits(CCPs) stained with the Alexa647/Cy3 in BS-C-1 cells[54-55]
图 13花青染料(35~40)分子式和假设的闪烁机制
Figure 13.Chemical structures of cyanine dyes(35~40) and the proposed photoblinking mechanism
图 14STORM荧光探针41~48结构式及细胞微管的超分辨图像[62]；Si-罗丹明荧光分子(49~55)衍生物以及分子54在合适pH环境中闪烁机制的模型及相应的化学结构式[65]; ATTO655标记的CCR5在水和重水不同环境下，分子56和57在光照时互相转化过程以及亚细胞结构图像[66]
Figure 14.Chemical structures of fluorescent dyes(41-48) used in STORM[62]; Chemical structures of 49-55 and proposed photoblinking mechanism of 54 in a suitable pH environment[65]; Photoswitching between 56 and 57 upon photoirradiation in the presence of oxygen and ATTO655-labelled CCR5 imaging of live cells in H2O and D2O[66]
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