Magnify is a universal molecular anchoring strategy for expansion microscopy

Abstract

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.

Current Progress in Expansion Microscopy: Chemical Strategies and Applications

“…For instance, by expanding samples 11-fold, Klimas et al demonstrated a lateral resolution of ∼15 nm when combining SOFI with Magnify (see section ). Under this resolution, the authors demonstrated that Magnify-SOFI can clearly resolve fine details of cilia and basal bodies in human stem-cell-derived lung organoids, similar as features previously demonstrated with electron microscopy.…”

“…(right) The isolated NHS and synaptophysin channels (top) and a magnified view of boxed region (bottom). Adapted with permission from ref . Copyright 2023 Springer Nature under CC BY 4.0.…”

“…Recently, Klimas et al reported molecule anchorable gel-enabled nanoscale fluorescence microscopy (Magnify) as a simple and convenient anchoring method for proteins, lipids, and nucleic acids without requirements of a separated anchoring step nor custom-synthesized anchors for different biomolecules (Figure b). , In this strategy, methacrolein was applied in the monomer solution to directly graft biomolecules to the hydrogel during the polymerization process. Through optimizing the homogenization step in a heated, denaturant-rich solution, the authors demonstrated that various biomolecules (e.g., proteins, lipids, and nucleic acids) can be retained at a high level, enabling postexpansion staining in ExM (see section ).…”

“…Given the wide variety of polymer architectures and chemical and physical considerations, the polymer is a defining component of any ExM experiment. Since the publishing of the original ExM protocol, various recipes have been explored to optimize ExM, e.g., enhance imaging resolution ,− or improve isotropy . Based on the mechanism of gel formation, polymerization can be divided into radical-induced polymerization or click-reaction-based polymerization.…”

“…For instance, the monomer solution needs to be thoroughly purged with N 2 many times to eliminate oxygen before performing polymerization. Another drawback is that this gel is compatible with only thin tissues (<50 μm). , …”