post-expansion biomolecule profiling of the digested and expanded tissue

Expansion Microscopy for Robust 11-Fold Expansion

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).

Author Spotlight: Universal Molecular Retention with 11-Fold Expansion Microscopy  

Universal Molecular Retention with 11-Fold Expansion Microscopy

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  

To begin, take the digested and expanded archived or fresh clinical tissue sample and proceed with antibody staining by placing it in a single well of a six-well cell culture plate. Wash the sample three times with PBS for 10 minutes each at room temperature. Depending on the sample size, add 0.5 to 2 milliliters of the primary antibodies in the staining buffer to cover the gel adequately.Incubate overnight at 37 degrees Celsius. Then, wash the sample three times with PBS for 10 minutes each at room temperature. Add the corresponding secondary antibodies and incubate for three hours at 37 degrees Celsius in the staining buffer of choice.Rewash the sample as demonstrated earlier, and add 1 to 2 milliliters of polyethylene glycol onto the sample in a six-well plate. Stain the sample with Fluor 4 conjugated NHS ester for three hours at room temperature. At the end of the incubation, wash the sample three times with PBS.Next, to perform lipid staining, take the expanded tissue sample washed three times in PBS, apply a conventional lipophilic dye diluted 200 fold in 2 milliliters of 0.1%Triton X-100 or PBS for 72 to 96 hours at room temperature, and wash at least three times with PBS. To perform FISH, prepare the hybridization buffer by combining 2 x SSC, 10%dextran sulfate, 20%ethylene carbonate, and 0.1%Tween 20. Place homogenized gel samples in a hybridization buffer preheated at 60 degrees Celsius for 30 minutes.Then, incubate the gel samples with a hybridization buffer containing 10 picomolar FISH probes against the target gene overnight at 45 degrees Celsius. Wash the samples with stringency wash buffer twice for 15 minutes each at 45 degrees Celsius and twice for 10 minutes at 37 degrees Celsius. Wash the sample three times with PBS for 10 minutes and proceed with imaging or storing the sample in PBS plus 0.02%sodium azide at 4 degrees Celsius.To fully expand and image the tissue, move the samples expanded fourfold in PBS to a glass bottom six-well imaging plate. If the sample has been expanded further, cut it into smaller pieces. Apply 0.1%polylysine to the glass surface.Place the sample on the slide, and remove any excess liquid around the gel with a paper laboratory cloth to prevent gel movement during imaging. Afterward, perform fluorescence imaging using a conventional wide-field microscope, a confocal microscope, or another imaging system of choice. After imaging, shrink the samples in PBS with at least three 10-minute washes, as long-term storage in deionized water leads to degradation of the fluorescent signal.Finally, store the samples in PBS with 0.02%sodium azide at 4 degrees Celsius. Confocal images of fully expanded mouse brain tissue allowed the visualization of proteins and cell and mitochondrial membrane structures. The nucleic acids of formal and fixed paraffin-embedded human lymph node tissue were also identified.After a 3.5-fold expansion of the kidney section, the crack-free expanded glomerulus was seen. However, inadequate anchoring or homogenization resulted in cracking, distortions, and the loss of labeled targets in another kidney section.

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Research

JoVE Journal

Biology

3.7K visualizações.  Carnegie Mellon University. Para começar, pegue a amostra de tecido clínico digerido e expandido arquivado ou fresco e prossiga com a coloração de anticorpos colocando-a em um único poço de uma placa de cultura de células de seis poços. Lavar a amostra três vezes com PBS durante 10 minutos cada à temperatura ambiente. Dependendo do tamanho da amostra, adicionar 0,5 a 2 mililitros dos anticorpos primários no tampão de coloração para cobrir adequadamente o gel.Incubar durante a noite a 37 graus Celsiu...