Nanoscale Imaging of Biomolecules using Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence Microscopy

Abstract

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.

Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

“…To fully demonstrate the potential of expansion SRS imaging, we chose a new ExM strategy that achieves a favorable balance between biomolecule retention, expansion factor, and sample generality 30 . The new method is called Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY) 30 .…”

“…To fully demonstrate the potential of expansion SRS imaging, we chose a new ExM strategy that achieves a favorable balance between biomolecule retention, expansion factor, and sample generality 30 . The new method is called Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY) 30 . To demonstrate its utility for SRS imaging, we first quantified the protein retention rate by imaging CH3 stretching vibration of proteins at 2941 cm -1 in paraformaldehyde (PFA)-fixed mouse kidney tissue pre-and post-expansion (Fig.…”

“…MAGNIFIERS relied on a reformulated hydrogel (Fig. 1a) which allows isotropic physical expansion up to 10-fold in a single round 30 . To theoretically estimate the improved spatial precision, we calibrated the resolution of the current SRS microscopy by imaging single 100-nm polystyrene beads at 3056 cm -1 (Supplementary Fig.…”

“…The MAGNIFY framework is known to retain biomolecules other than proteins such as lipids and nucleic acids 30 . Thus, MAGNIFIERS holds great promise for label-free chemically-defined nanoscale imaging in a comprehensive view.…”

“…Recently, a new ExM framework has emerged that offers larger expansion factors along with broad biomolecule retention and tissue compatibility 30 , capable of expanding a multitude of tissue types with standard fixation, from PFA-fixed mouse brain and human lung organoid to an array of clinically relevant formalin-fixed paraffin-embedded (FFPE) human tissues which have proved challenging to expand in the past. By combining SRS imaging with this framework, we lay out an approach named Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS).…”