Magnify is a universal molecular anchoring strategy for expansion microscopy

Understanding biological systems requires precise knowledge of spatial arrangements of components at various scales. Expansion microscopy (ExM) enables nanoscale imaging using standard fluorescent microscopes by physically magnifying preserved specimens embedded in a swellable hydrogel. Existing ExM protocols typically involve a prior anchoring step using reactive chemicals to link specific labels and biomolecule classes to the gel. This step adds complexity and can limit the range of compatible biomolecules and tissue types. The need for an improved ExM protocol that is user-friendly, provides high expansion factors with minimal distortion, conserves diverse biomolecules, and is compatible with various tissue types and fixation methods remains. This study aims to address these limitations.

Expansion microscopy has emerged as a powerful technique for nanoscale imaging, allowing researchers to visualize subcellular structures with conventional microscopes. Several ExM protocols have been developed, focusing on imaging proteins, nucleic acids, and lipids. However, these protocols often suffer from limitations such as low expansion factors (typically around fourfold in tissues), reliance on specialized and commercially unavailable anchoring agents, and incompatibility with certain tissue types or fixation methods. High expansion factor methods (greater than tenfold) have primarily been demonstrated in cultured cells and brain tissue sections, leaving many tissue types limited to approximately 70nm resolution. The need for a more broadly applicable and easily adoptable expansion method, capable of achieving high expansion factors while preserving a wide range of biomolecules, is apparent.

Magnify eliminates the need for a separate anchoring step by incorporating methacrolein, a small molecule used in classical fixation protocols, into the hydrogel monomer solution. Methacrolein reacts with biomolecules during gelation, anchoring them to the gel matrix without requiring pre-treatment. The authors used a mechanically robust hydrogel formulation consisting of DMAA, SA, AA, and Bis, capable of achieving 8.5-fold expansion in FFPE human kidney tissue sections and up to 11-fold expansion in freshly preserved mouse brain slices after heat denaturation. The protocol includes a homogenization step using a hot, denaturant-rich solution to improve expansion and biomolecule retention. Various tissues (mouse brain, human kidney (FFPE), human lung organoids) were processed using Magnify, and subsequent staining (pre- and post-expansion) was performed with antibodies, fluorescent dyes (including lipid-specific dyes), and DNA/RNA FISH probes. Super-resolution optical fluctuation imaging (SOFI) was combined with Magnify to further enhance resolution. Detailed protocols for tissue preparation, in situ polymerization, homogenization, staining (pre- and post-expansion), and SOFI imaging and analysis are provided in the supplementary information.

Magnify successfully achieves a linear expansion factor of up to 11x, resulting in an effective resolution of approximately 25 nm with a conventional microscope (280 nm diffraction-limited objective) and approximately 15 nm resolution when combined with SOFI. The method showed high retention of proteins, nucleic acids, and lipids, enabling post-expansion labeling. Magnify successfully imaged various specimens, including synaptic proteins in mouse brain, podocyte foot processes in FFPE human kidney, and ciliary structures in human lung organoids. The method proved compatible with different tissue types and fixation methods, including FFPE samples, which are notoriously challenging to expand while preserving epitopes. The use of a hot denaturant-rich solution during homogenization was crucial for effective expansion and biomolecule preservation in FFPE samples, and improved antibody accessibility compared to conventional antigen retrieval. Magnify also enabled nanoscale observation of lipid membrane structures, revealing details such as mitochondrial cristae and the ultrastructure of lipids within neuronal processes. Postexpansion DNA FISH was successfully demonstrated, enabling super-resolution imaging of nucleic acids. Endogenous fluorescent proteins were successfully imaged in transgenic mouse brain tissue. Magnify combined with SOFI (Magnify-SOFI) achieved sub-20 nm resolution, allowing for detailed visualization of ciliary structures and their defects in human lung organoids treated with paclitaxel or derived from patients with CCDC39 mutations. Quantifications comparing Magnify-SOFI with electron microscopy showed consistent results in identifying ciliary defects.

Magnify addresses several key limitations of existing ExM methods. Its simplicity and versatility make it a broadly applicable tool for nanoscale imaging. The elimination of the separate anchoring step simplifies the protocol and broadens the range of compatible biomolecules. The high expansion factor and compatibility with various tissue types, including FFPE samples, expand its applicability to a wider range of research questions and clinical samples. The combination with SOFI significantly enhances resolution, approaching the capabilities of specialized super-resolution microscopy without requiring specialized equipment or fluorophores. The ability to preserve and image diverse biomolecules, including lipids and nucleic acids, opens new avenues for studying various biological processes and disease mechanisms. The successful application of Magnify to study ciliopathies demonstrates its potential for advancing our understanding of human diseases.

Magnify represents a significant advancement in expansion microscopy, providing a simple, versatile, and high-resolution approach for nanoscale imaging. Its compatibility with various biomolecules, tissue types, and fixation methods, coupled with its ability to achieve sub-20 nm resolution when combined with SOFI, makes it a powerful tool for various biological and biomedical research applications. Future research may explore its application to even thicker tissues, whole organs, and other imaging modalities.

While Magnify demonstrates high performance across various samples, there is some inter-class variation in expansion factors depending on tissue type, methacrolein concentration, and homogenization time. The use of a hot denaturant-rich solution, although crucial for expansion, may lead to some loss of fluorescent protein signal. The study mainly focused on specific biomolecules and combinations; further investigation may be needed to fully explore the range of compatible biomolecules and their interactions within the Magnify framework.