universal-molecular-retention-with-11-fold-expansion-microscopy

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 ...

Carnegie Mellon University

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel-enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label-free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy.

Expansion microscopy enables nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a crosslinked water-swellable hydrogel. Current expansion microscopy protocols require prior treatment with reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. We describe a strategy called Magnify, which uses a mechanically sturdy gel that retains nucleic acids, proteins and lipids without the need for a separate anchoring step. Magnify expands biological specimens up to 11 times and facilitates imaging of cells and tissues with effectively around 25-nm resolution using a diffraction-limited objective lens of about 280 nm on conventional optical microscopes or with around 15 nm effective resolution if combined with super-resolution optical fluctuation imaging. We demonstrate Magnify on a broad range of biological specimens, providing insight into nanoscopic subcellular structures, including synaptic proteins from mouse brain, podocyte foot processes in formalin-fixed paraffin-embedded human kidney and defects in cilia and basal bodies in drug-treated human lung organoids.

Magnify is a universal molecular anchoring strategy for expansion microscopy.

Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. We developed µMagnify, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. We formulated an enzyme cocktail specifically designed for robust cell wall digestion and expansion of microbial cells without distortion while efficiently retaining biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. Additionally, we developed an associated virtual reality tool to facilitate the visualization and navigation of complex three-dimensional images generated by this method in an immersive environment allowing collaborative exploration among researchers around the world. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables development of new diagnosis strategies against infectious diseases.

MicroMagnify: a multiplexed expansion microscopy method for pathogens and infected tissues.

A New Expansion Microscopy Method Optimized for Microbiology.

11-fold Expansion Microscopy with Universal Molecular Retention Using Magnify.

Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus-infected mouse tone, and formalin-fixed paraffin-embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

Zhangyu Cheng

Department of Biological Sciences

Carnegie Mellon University

Universal Molecular Retention with 11-Fold Expansion Microscopy