Name: Sample Digestion and Tissue Expansion of Polymerized Tissue
Uploaded: 2023-10-06T12:20:00
Description: Watch this Scientific Journal Video about Sample Digestion and Tissue Expansion of Polymerized Tissue at JoVE.com

sample digestion and tissue expansion of polymerized tissue

堅牢な11倍拡張のための拡張顕微鏡

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).

著者スポットライト:11倍拡大顕微鏡による普遍的な分子保持  

11倍膨張顕微鏡によるユニバーサル分子保持

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 Sample Digestion and Tissue Expansion of Polymerized Tissue at JoVE.com

Sample Digestion and Tissue Expansion of Polymerized Tissue  

重合組織のサンプル消化と組織拡大

まず、重合したアーカイブされた新鮮な組織またはパラホルムアルデヒド固定マウスの脳組織でスライドを取ります。目の保護具を着用し、かみそりの刃を使用してゲル化チャンバーの2つのスライドガラスを分離します。組織の周りのブランクゲルをトリミングして体積を最小限に抑え、非対称に切断して均質化後のゲルの配向を追跡します。かみそりの刃でスライドからゲルを含む組織をそっと持ち上げます。それを2ミリリットルの遠沈管に移します。摂氏80度に予熱したホモジナイズバッファーでチューブの上部を満たします。次に、摂氏80度で8〜60時間振とうしながらサンプルをインキュベートします。遠沈管の内容物を6ウェルプラスチック細胞培養プレートの1ウェルに注ぎ、トランスファーピペットで変性バッファーを除去します。ヒドロゲルから残りのSDSを完全に除去するには、1%非イオン性界面活性剤溶液でサンプルを洗浄します。最後に、サンプルをPBSと0.02%アジ化ナトリウム(摂氏4度)で保存します。

Research

JoVE Journal

Biology

Sample Digestion and Tissue Expansion of Polymerized 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 ...