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

견고한 11배 팽창을 위한 팽창 현미경

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).

저자 스포트라이트: Universal Molecular Retention with 11-Fold Expansion Microscopy  

Universal Molecular Retention with 11-Fold Expansion Microscopy(11배 팽창 현미경을 통한 범용 분자 유지)

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  

중합된 조직의 시료 분해 및 조직 확장

시작하려면 중합 된 보관 된 신선한 조직 또는 파라 포름 알데히드 고정 마우스 뇌 조직으로 슬라이드를 가져 가십시오. 보안경을 착용하고 면도날을 사용하여 겔화 챔버의 두 개의 유리 슬라이드를 분리합니다. 부피를 최소화하기 위해 조직 주위의 블랭크 젤을 다듬고 균질화 후 젤의 방향을 추적하기 위해 비대칭으로 절단해야 합니다.면도날로 슬라이드에서 젤이 함유된 티슈를 부드럽게 들어 올립니다. 2밀리리터 원심분리기 튜브로 옮깁니다. 섭씨 80도로 예열된 균질화 버퍼로 튜브를 상단까지 채웁니다.그런 다음 섭씨 80도에서 8-60시간 동안 진탕하면서 샘플을 배양합니다. 원심분리기 튜브의 내용물을 6웰 플라스틱 세포 배양 플레이트의 단일 웰에 붓고 이송 피펫으로 변성 완충액을 제거합니다. 하이드로겔에서 남은 SDS를 완전히 제거하려면 1% 비이온성 계면활성제 용액으로 샘플을 세척합니다.마지막으로 샘플을 섭씨 4도에서 PBS와 0.02% 아지드화나트륨에 보관합니다.

Research

JoVE Journal

Biology

Sample Digestion and Tissue Expansion of Polymerized Tissue

Expansion Microscopy for Robust 11-Fold Expansion

Universal Molecular Retention with 11-Fold Expansion Microscopy

Author Spotlight: 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 ...