Expansion microscopy (ExM) is a powerful imaging strategy that offers a low-cost solution for nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a cross-linked water-swellable hydrogel. Current ExM protocols require prior treatment with specialized reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. In addition, most techniques reportedly use strong Proteinase K to digest endogenous epitopes to enable expansion and are limited by using mechanically fragile gel formulas to expand specimens by at most 4.5× linearly. Here we describe a new ExM framework, Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY), that uses a mechanically sturdy gel that enables broad retention of nucleic acids, proteins, and lipids without the need for a separate anchoring step. MAGNIFY expands biological specimens up to 11× and facilitates imaging of cells and tissues with effectively ~25-nm-resolution using an ∼280-nm diffraction-limited objective lens on conventional optical microscopes or with ~13 nm-resolution if combined with Super-resolution Optical Fluctuation Imaging (SOFI). Further, MAGNIFY generalizes well across a broad range of biological specimens, providing insight into nanoscopic subcellular structures including synaptic proteins from mouse brain, podocyte foot processes in human kidney, and defects in cilia and basal bodies in drug-treated human lung organoids. MAGNIFY provides a novel advance that expands the precision, utility, accessibility, and generality of subcellular nanoscopy.

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.

Through targeted binding to the cell membrane, structural DNA nanotechnology has the potential to guide and affix biomolecules such as drugs, growth factors and nanobiosensors to the surfaces of cells....

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.