Name: Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue
Uploaded: 2023-10-06T12:20:00
Description: Watch this Scientific Journal Video about Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue at JoVE.com

post-expansion biomolecule profiling of the digested and expanded tissue

用于稳健 11 倍扩增的扩增显微镜

Biological systems&#160;exhibit structural heterogeneity, from the limbs and the organs down to the levels of proteins at the nanoscale. Therefore, a complete understanding of the operation of these systems requires visual examination across these size scales. However, the diffraction limit of light causes challenges in visualizing structures smaller than ~200-300 nm on a conventional fluorescence microscope. In addition, optical super-resolution methods1,2,3, such as stimulated emission depletion (STED), photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM), though powerful, present their own challenges, as they require expensive hardware and reagents and often have slow acquisition times and a poor ability to image large volumes in 3D.
Expansion microscopy4 (ExM) provides an alternative means of circumventing the diffraction limit of light by covalently anchoring biomolecules into a water-swellable polymer gel and physically pulling them apart, thus rendering them resolvable on conventional optical microscopes. A multitude of ExM protocol variants have been developed since the original publication of&#160;ExM less than a decade ago, and these protocols allow the direct incorporation of proteins5,6,7, RNA8,9,10, or lipids11,12,13 into the gel network by altering the chemical anchor or expanding the sample further (thus improving the effective resolution) either in a single step14 or multiple iterative steps15,16. Until recently, no single ExM protocol could retain these three biomolecule classes with a single commercially available chemical anchor while providing a mechanically sturdy gel that could expand ~10-fold in a single expansion round.
Here, we present Magnify17, a recent addition to the ExM arsenal that uses methacrolein as the biomolecule anchor. Methacrolein forms covalent bonds with tissue like that of paraformaldehyde, ensuring that multiple classes of biomolecules can be retained within the gel network without requiring various specific or custom anchoring agents. Additionally, this technique can expand a broad spectrum of tissues up to 11-fold, including notoriously challenging samples such as formalin-fixed paraffin-embedded (FFPE) clinical samples. Previous methods for expanding such mechanically rigid samples required harsh protease digestion, rendering the antibody labeling of proteins of interest impossible after the sample had been expanded. In contrast, this technique achieves the expansion of FFPE clinical samples using a hot denaturing solution, thus preserving whole protein epitopes within the gel, which can be targeted for post-expansion imaging (Figure 1).

作者聚焦：使用 11 倍扩增显微镜进行通用分子截留  

采用 11 倍扩增显微镜的通用分子保留

We're trying to find easy and low cost ways for researchers to image their biological specimens at resolutions below the diffraction limit, particularly in the areas of neuroscience and clinical pathology. Many recent expansion microscopy developments involve modifying gel chemistry to expand samples further, changing the anchoring molecule to visualize different targets and utilizing heating alteration to preserve protein epitopes. With innovated magnify, a robust expansion microscopy method that allows nanoscale imaging of various biological or clinical specimens.It simplifies super resolution microscopy by eliminating specialized equipment needs while preserving intricate biological features with up to 15 nanometers resolution, hence increasing accessibility and utility in labs worldwide. Previously users had to experiment with different expansion microscopy techniques, depending on the tissue and biomolecules they wanted to image. Magnify enables high resolution imaging across various biomolecules and tissue types without requiring specific anchoring steps or specialized equipment, thus bridging a significant gap in nanoscale imaging.Magnify expands tougher clinical samples over fourfold while preserving protein epitopes enabling more accurate investigation of queuing disease. Its chemical anchor also preserves a wide range of biomolecules, making it useful for basic research in animal models Magnify simplifies nanoscale imaging of biomolecules and intact specimens without complex optics or specialized equipment. It's adaptable across various imaging modalities and expansion microscopy strategies, allowing accessible cost-effective nanoscale imaging that may accelerate drug treatments and disease studies while potentially transforming tissue atlas generation.Magnify offers endless possibilities for research. Current focus areas include expanding tissue site, investigating pathological human samples at the nanoscale and studying the smallest scale changes on brain during learning and disease.

Watch this Scientific Journal Video about Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue at JoVE.com

Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue  

消化和扩增组织的扩增后生物分子分析

首先，取消化和扩增的存档或新鲜临床组织样品，并将其置于六孔细胞培养板的单孔中进行抗体染色。用PBS洗涤样品三次，每次在室温下洗涤10分钟。根据样品大小，在染色缓冲液中加入0.5至2毫升一抗，以充分覆盖凝胶。在 37 摄氏度下孵育过夜。然后，在室温下用PBS洗涤样品三次，每次10分钟。加入相应的二抗，并在所选的染色缓冲液中以37摄氏度孵育3小时。如前所述重新洗涤样品，并在六孔板中向样品中加入1至2毫升聚乙二醇。在室温下用Fluor 4偶联NHS酯对样品染色三小时。在孵育结束时，用PBS洗涤样品三次。接下来，为了进行脂质染色，取在PBS中洗涤三次的膨胀组织样品，在2毫升0.1%Triton X-100或PBS中稀释200倍的常规亲脂性染料在室温下72至96小时，并用PBS洗涤至少三次。为了进行FISH，通过混合2 x SSC，10%葡聚糖硫酸盐，20%碳酸乙烯酯和0.1%吐温20来制备杂交缓冲液。将匀浆凝胶样品置于60摄氏度预热的杂交缓冲液中30分钟。然后，将凝胶样品与含有10皮摩尔FISH探针的杂交缓冲液在45摄氏度下孵育过夜。用严格的洗涤缓冲液洗涤样品两次，每次 45 摄氏度下 15 分钟，在 37 摄氏度下洗涤两次，每次 10 分钟。用PBS洗涤样品3次10分钟，然后继续成像或将样品储存在PBS加0.02%叠氮化钠中，温度为4摄氏度。为了完全扩增和成像组织，将PBS中扩增四倍的样品移动到玻璃底六孔成像板上。如果样品已进一步扩展，请将其切成小块。将0.1%聚赖氨酸涂在玻璃表面。将样品放在载玻片上，并用实验室纸布去除凝胶周围的任何多余液体，以防止凝胶在成像过程中移动。然后，使用传统的宽视场显微镜、共聚焦显微镜或其他选择的成像系统进行荧光成像。成像后，将样品在PBS中收缩至少三次10分钟的洗涤，因为长期储存在去离子水中会导致荧光信号降解。最后，将样品储存在含有0.02%叠氮化钠的PBS中，温度为4摄氏度。完全扩增的小鼠脑组织的共聚焦图像允许蛋白质以及细胞和线粒体膜结构的可视化。还鉴定了正式和固定石蜡包埋的人淋巴结组织的核酸。肾切片扩张3.5倍后，可见无裂缝扩张的肾小球。然而，锚定或均质化不足导致开裂、变形和另一个肾脏切片中标记靶标的丢失。

Research

JoVE Journal

Biology

Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue

Universal Molecular Retention with 11-Fold Expansion Microscopy

The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been ...