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

Expansionsmikroskopi för robust 11-faldig 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).

Författare i fokus: Universell molekylär retention med 11-faldig expansionsmikroskopi  

Universell molekylär retention med 11-faldig expansionsmikroskopi

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  

Biomolekylprofilering efter expansion av den spjälkade och expanderade vävnaden

För att börja, ta det smälta och expanderade arkiverade eller färska kliniska vävnadsprovet och fortsätt med antikroppsfärgning genom att placera den i en enda brunn på en sexbrunnscellodlingsplatta. Tvätta provet tre gånger med PBS i 10 minuter vardera vid rumstemperatur. Beroende på provstorleken, tillsätt 0,5 till 2 ml av de primära antikropparna i färgningsbufferten för att täcka gelén tillräckligt.Inkubera över natten vid 37 grader Celsius. Tvätta sedan provet tre gånger med PBS i 10 minuter vardera vid rumstemperatur. Tillsätt motsvarande sekundära antikroppar och inkubera i tre timmar vid 37 grader Celsius i valfri färgningsbuffert.Tvätta provet igen enligt tidigare och tillsätt 1 till 2 ml polyetylenglykol på provet i en sexbrunnsplatta. Färga provet med fluor 4-konjugerad NHS-ester i tre timmar vid rumstemperatur. Vid slutet av inkubationen tvättas provet tre gånger med PBS.Därefter, för att utföra lipidfärgning, ta det expanderade vävnadsprovet tvättat tre gånger i PBS, applicera ett konventionellt lipofilt färgämne utspätt 200 gånger i 2 ml 0,1% Triton X-100 eller PBS i 72 till 96 timmar vid rumstemperatur och tvätta minst tre gånger med PBS. För att utföra FISH, förbered hybridiseringsbufferten genom att kombinera 2 x SSC, 10% dextransulfat, 20% etylenkarbonat och 0,1% Tween 20. Placera homogeniserade gelprover i en hybridiseringsbuffert som förvärmts vid 60 grader Celsius i 30 minuter.Inkubera sedan gelproverna med en hybridiseringsbuffert innehållande 10 picomolära FISH-sonder mot målgenen över natten vid 45 grader Celsius. Tvätta proverna med stringenstvättbuffert två gånger i 15 minuter vardera vid 45 grader Celsius och två gånger i 10 minuter vid 37 grader Celsius. Tvätta provet tre gånger med PBS i 10 minuter och fortsätt med avbildning eller lagring av provet i PBS plus 0,02% natriumazid vid 4 grader Celsius.För att helt expandera och avbilda vävnaden, flytta proverna expanderade fyrfaldigt i PBS till en glasbotten med sex brunnar. Om provet har expanderats ytterligare, skär det i mindre bitar. Applicera 0,1% polylysin på glasytan.Placera provet på objektglaset och avlägsna överflödig vätska runt gelén med en papperslaboratorieduk för att förhindra gelrörelse under avbildning. Utför därefter fluorescensavbildning med hjälp av ett konventionellt bredfältmikroskop, ett konfokalmikroskop eller ett annat bildsystem som du väljer. Efter avbildning, krympa proverna i PBS med minst tre 10-minuters tvättar, eftersom långvarig lagring i avjoniserat vatten leder till nedbrytning av den fluorescerande signalen.Slutligen förvara proverna i PBS med 0,02% natriumazid vid 4 grader Celsius. Konfokala bilder av fullt expanderad hjärnvävnad från möss möjliggjorde visualisering av proteiner och cell- och mitokondriella membranstrukturer. Nukleinsyrorna i formell och fast paraffininbäddad human lymfkörtelvävnad identifierades också.Efter en 3,5-faldig expansion av njursektionen sågs den sprickfria expanderade glomerulusen. Otillräcklig förankring eller homogenisering resulterade emellertid i sprickbildning, snedvridningar och förlust av märkta mål i en annan njursektion.

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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 ...