由于光的衍射，传统的光学显微镜具有不同的限制，因此，它无法解析相距不到200 nm的功能。
Microscopy technique development has historically focused on improvements in imaging techniques, facilitating the resolution of individual molecules. The beginning of the 21英石century saw the development of super-resolution microscopy, allowing imaging at a resolution beyond the 200 nm limit.7,8
然而，不幸的是，即使是超分辨率技术也有局限性，包括有限的成像深度，有限的视野，与多色实验的不兼容，高能量照明强度要求以及缓慢的获取时间。
为了解决这些问题，少数创意研究小组朝着不同的方向发展，探索了直接，标准化的纳米级成像的方法。这项工作的结果之一是扩展显微镜（EXM） - 一种成像协议，它使传统的光显微镜能够解决以前无法区分的密集包装或亚分式限制（<200 nm）细节。
爱德华·博伊登（Edward Boyden）在麻省理工学院（MIT）的团队报告了2015年这种新的放大倍率的发展。这是在该团队试图克服大脑大规模神经回路分子时的挑战时开发的。博伊登的研究小组成功地开发了一种放大样品本身的方法，而不是放大样品发射的信号。2
Table 1.词汇表。
| 词汇表： | |
|---|---|
| ACX | Acryoyl -X，SE，6-（丙烯酰氨基己糖）酯琥珀酰基酯（一种与蛋白质反应并在聚丙烯酰胺基质中进行共聚的交联剂） |
| Diexm | 差分扩展显微镜 |
| DMAA | N，N二甲基氨酰胺酸 - （非离子丙烯酸单体，可以使膨胀的聚合物颗粒）yabo214 |
| ExFish | 膨胀显微镜fluorescence in situ hybridization |
| ExM | 膨胀显微镜 |
| FP | 荧光蛋白 |
| GA | glutharaldehyde (crosslinking fixative, penetrate membran more slowly than PFA) |
| HCR | hybridization chain reaction |
| iExM | 迭代膨胀显微镜 |
| 如果 | 免疫荧光团 |
| MA NHS |
methacrylic acid N-hydroxysuccinimide ester (crosslinking agent that reacts with amines of proteins and can be copolymerized in polyacrylamide matrices) |
| 地图 | maximum analysis of the proteome |
| MBAA or BA |
N’N Methylenebis (acrylamide) /(bisacrylamide) crosslinking agent that polymerize with acrylamide and creates crosslinks within the polyacrylamide gel) |
| PFA | paraformaldehyde (crosslinking fixative, preserves the secondary/tertiary structures of proteins) |
| ProExM | 蛋白质的扩张显微镜 |
| SDS | 十二烷基硫酸钠（阴离子表面活性剂可用于结实和解离蛋白质） |
| SRRF | super resolution radial fluctuations |
图1。HeLa细胞的共聚焦图像非扩展的微管（右）和4.5倍线性扩展的微管（左）。Imaging was performed on an Andor spinning-disk confocal microscope (Dragonfly) with a 40×, numerical aperture (NA) 1.15 water- immersion objective.（A）HeLa细胞的共聚焦图像在细胞底部的单个XY平面上成像。左上方的插图放大在右中间的小盒子上。（b）Confocal image of a ∼4.5× linearly expanded HeLa cell with immunostained microtubules imaged at a single XY plane at the bottom of the cell. The inset in the upper left zooms in on the small box at the bottom left. Scale bars in (B) indicate post-expansion scales. Only a fraction of an expanded cell fills the entire field of view. The respective insets display a zoom of the respective small boxes of the full field of view. (Zhang, C., et al, Current Protocols in Neuroscience, 2020). Image Credit: Andor Technology Ltd.
ExM is highly cost-effective, and this sample preparation method involves synthesizing a dense interconnected web of swellable polymer within the biological specimen of interest. The tissue inside the polymer matrix can be expanded and labeled and then when immersed in water, this expansion isotropically pulls apart the cellular structures, resulting in large gaps between each biomolecule.
各向同性的样品被放大，这意味着可以通过标准显微镜实现有效的更高分辨率。在纯水中报道了4倍线性扩展，这意味着体积膨胀的膨胀。在扩展的微管HeLa细胞样品上采用Andor Spinning Disk共聚焦（Dragonfly）样品（图1）说明了扩展显微镜协议的使用如何揭示样品中以前看不见的特征。
Extra care should be taken when preparing expanded samples, however, and successfully implementing expansion microscopy protocols relies on a number of considerations:
Embedding safely - a non-invasive methodology must be used to insert the polymer into cells. This is achieved by carefully inserting small monomers into the cells and tissues - these monomers are building blocks of the hydrogel that will be expanded, so the monomer polymerization must only be triggered once these are fully inside the preserved cells and tissues.
扩展而无需取消结构 - 必须扩展样品，同时维持细胞的结构组织。为了确保样品在膨胀过程中不会变形，用热/洗涤剂或酶消化处理样品，以机械地将样品均匀化，以确保组织保持完整。
Table 2.扩展显微镜方案的比较。有关缩写，请参见上面的词汇表。
| Protocol name | ExM （博伊登实验室） |
EXM（沃恩实验室） | ProExM (Boyden 实验室） |
地图 (Chung 实验室） |
Exfish（博伊登实验室） |
|---|---|---|---|---|---|
| 水凝胶 | 丙烯酰胺 +丙烯酸钠+++ MBAA | 丙烯酰胺 +丙烯酸钠+++ | 丙烯酰胺 +丙烯酸钠+++ | 丙烯酰胺+++丙烯酸钠 + ba | 丙烯酰胺丙烯酸钠MBAA |
| 链接 代理人 |
acrydite | MA-NHS GA | Acryloyle-X（ACX） | 多聚甲醛丙烯酰胺 | LabelX (AcX + Label-IT amine) |
| Disruption agent | proteinase K | proteinase K | proteinase K | SDS | proteinase K |
| Disruption type | 消化 | 消化 | 消化or gentle disruption | denaturation dissociation | 消化 |
| 扩展因子（线性） | 4。5 | 4.0-4.2 | 4 | 4 | 3.3 |
| Resolution | 70 nm | 65nm | 70 nm | 60 nm | / |
| FP preservation | no | yes | 是（强度为50％） | no | no |
| 如果英石aining | no | yes | yes | yes | no |
| Sample | cells, brain tissue | cells, brain tissue | cells, brain, pancreas, lung, spleen tissues | cells, brain, spinal cord, lung, heart, liver, kidney, intestine tissues | cells, brain tissue |
| 目标 | 蛋白质 | 蛋白质和DNA | 蛋白质 | 蛋白质和糖糖 | RNA和DNA |
| Pros comments | 第一个方法报告了 | 常规荧光团细胞器水平 | 常规荧光团后曝光标记 | post-expansion labeling whole organ level multiplexed staining | 3D成像膨胀后鱼类多路复用和HCR扩增 |
| Cons comments | complex protocol no standard fluorophores | 膨胀前标记仅荧光损失 | incomplete homogenization fluorescence loss | 不兼容 w/FPs preparation lost after 3days |
/ |
| 参考 | Chen等，2015 Science亚博老虎机网登录 | Chozonski et al. 2016 Nature Methods | Tillberg等，2016 Nature Biotech | Ku等，2016 Nature Biotch | Chen等人，2016年自然方法 |
| 变体 | iExM | x10 | x10;Diexm | UEXM |
图2。扩张前显微镜的原理。1）固定细胞2）通过在样品上进行免疫染色的标记，并将生物分子共价固定在凝胶3）凝胶过程中，将试样浸入单体溶液中，并形成化学网络。4）在均质化过程中，样品结构被酶促消化切碎，以确保组织保持完整。5）浸入水后，会自发扩张。在纯水中报道了4倍线性膨胀（64个体积膨胀）。图片来源：Andor Technology Ltd.
扩展显微镜有两种主要方法：
- Staining the biomolecules before expanding the tissue (Figure 2)
- Expanding the tissue before staining the biomolecules (Figure 3)
Figures 2 and 3 display protocols in which cells and tissues can be expanded up to 4x. The 200 nm diffraction limit of light dictates what can be distinguished as separated by light microscopes, and the linear expansion achieved is a factor of 4. Biomolecules that are separated up to 50 nm will be visible under the light microscope when using these protocols.
图3。膨胀后显微镜的原理1）细胞的固定2）内源性蛋白与凝胶的共价锚定为凝胶3）含有高聚丙烯酰胺浓度的水凝胶嵌入4）非跨链接蛋白的变性和解离蛋白5）在水中膨胀5）浸入后6）在多轮中进行免疫染色。图片来源：Andor Technology Ltd.
扩展显微镜的基本步骤
荧光信号还通过膨胀在各向同性上扩展，从而导致有效的更高分辨率。在下面和图2和3中概述了扩展显微镜协议的关键步骤。
- 标记 - 荧光团用于标记生物分子，尽管在膨胀后标记中将在膨胀后结束此步骤。
- Anchoring - biomolecules and/or labels are covalently fitted with molecular handles, and these cross-linkers will enable the polymer matrix to exercise force on the biomolecules.
- 凝胶化 - 在这里，聚合物链被分解成其组成部分或构件，以避免对细胞损坏。然后将试样浸入单体溶液（丙烯酸钠）和穿透性水凝胶（钠聚丙烯酸酯）中。一旦溶液和氢在细胞内部，就会触发化学反应，从而导致单体结合并形成所需聚合物的网络。
- Homogenization – this step aims to avert sample distortion and ensure that specimen organization is maintained. The sample will be chemically disrupted by enzymatic digestion or via a heat and detergent treatment (denaturation/dissociation). The homogenization treatment used will depend on the nature of the specimen and the specific molecules to be visualized.
- Expansion - the specimen is immersed in water, and the water diffuses into the polyelectrolyte hydrogel via osmotic force, promoting polymer expansion. The expansion will pull apart anchored biomolecules in an isotropic fashion, creating substantial gaps between individual biomolecules. The expanded specimen’s spatial organization is preserved, therefore allowing nanoscale imaging using standard fluorescent microscopes.
Figure 4.扩展有丝分裂细胞的图像。为小管蛋白（绿色）和DNA（蓝色）染色的分裂细胞的最大投影图像。使用后膨胀协议标记的单元格，以及用安多蜻蜓图像。图片来源：样本由约书亚·沃恩（Joshua C Vaughan）（华盛顿大学）提供。
Most Popular Protocols of Expansion Microscopy
自初始开发以来，已经发布了许多不同的扩展显微镜协议，目前有各种应用程序的变化和改进。下表总结了扩展显微镜的关键协议。
To summarize; two main strategies of expansion microscopy exist side by side: pre and post-expansion labeling strategies (Figures 2 and 3). These different protocols are adapted to certain cellular structures, so when beginning to use expansion microscopy imaging, appropriate care must be taken to select the most appropriate protocol for visualizing the subcellular structure in question.
It may be necessary to optimize a protocol in order to better suit experimental conditions, and researchers are advised to design proper experimental controls in order to demonstrate the isotropic expansion of the structure to be analyzed.
参考和进一步阅读
- Chang，J.-B。等。（2017）。“迭代膨胀显微镜。“Nature Methods 14(6): 593-599.
- Chen，F。等。（2015）。“膨胀显微镜。“Science 347(6221): 543-548.
- Chen，F。等。（2016）。“Nanoscale imaging of RNA with expansion microscopy。“Nature Methods 13(8): 679-684
- Chozinski，T。J.等。（2016）。“膨胀显微镜with conventional antibodies and fluorescent proteins。“Nature Methods 13(6): 485-488
- Gambarotto, D., et al. (2019). "Imaging cellular ultrastructures using expansion microscopy (U-ExM)。“自然方法16（1）：71-74
- Ku T，Swaney J，Park JY等。（2016年）”Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues。” Nat Biotechnol. 34 (9):973-981. doi:10.1038/nbt.3641
- Schermelleh, L., et al. (2019). "Super-resolution microscopy demystified。“Nature Cell Biology 21(1): 72-84.
- Schermelleh, L., et al. (2010). "超分辨率荧光显微镜指南。
- Truckenbrodt，S。等。(2018). "X10 expansion microscopy enables 25-nm resolution on conventional microscopes。“EMBO reports 19(9): e45836.
- Truckenbrodt，S。等。（2019）。“X10扩展显微镜优化的实用指南自然协议14（3）：832-863。
- Tillberg PW，Chen F，Piatkevich KD等。（2016）”Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies。“Nat Biotechnol. 34 (9):987-992.
- Wassie，A。T.等。（2019）。“扩展显微镜：生物学研究原理和用途。“自然方法16（1）：33-41
- Zhang, C., Kang, J. S., Asano, S. M., Gao, R., & Boyden, E. S. (2020) “膨胀显微镜for beginners: Visualizing microtubules in expanded cultured HeLa cells。” Current Protocols in Neuroscience
致谢
Produced from materials originally authored by Dr. Sébastien Bellow and Dr. Claudia Florindo from Oxford Instruments Andor.
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