Abstract
Fluorescence-based expansion microscopy (ExM) is a new technique which can yield nanoscale resolution of biological specimen on a conventional fluorescence microscope through physical sample expansion up to 20 times its original dimensions while preserving structural information. It however inherits known issues of fluorescence microscopy such as photostability and multiplexing capabilities, as well as an ExM-specific issue in signal intensity reduction due to a dilution effect after expansion. To address these issues, we propose using antigen-targeting plasmonic nanoparticle labels which can be imaged using surface-enhanced Raman scattering spectroscopy (SERS) and dark-field spectroscopy. We demonstrate that the nanoparticles enable multimodal imaging: bright-field, dark-field and SERS, with excellent photostability, contrast enhancement and brightness.
Highlights
Molecular labeling plays a crucial role in biomedical imaging, including microbiology, histopathology, and disease diagnosis
We demonstrate that these NPs are effective for histological labeling in standard fixed paraffin-embedded (FPE) tissue sections and have several features that make them well suited for expansion microscopy (ExM), namely increased photostability, high dark-field contrast and sensitivity to binding sites separation distances through plasmonic coupling effects
We demonstrate the feasibility of using plasmonic nanoparticle-based expansion microscopy with both SERS and dark-field spectroscopic imaging
Summary
Molecular labeling plays a crucial role in biomedical imaging, including microbiology, histopathology, and disease diagnosis. A recent alternative is expansion microscopy (ExM) [4,5,6,7], which circumvents the optical challenge by embedding the sample within a swellable polymer matrix that expands isotropically, allowing spatial features below the diffraction limit to become resolvable with non-SR imaging systems. While studies using this technique have been tested on a variety of tissue types [8], they inherit known issues of fluorescence microscopy such as photostability and multiplexing capabilities. While the effect of this step on target epitopes is not well understood, performing multi-pass multiplex labeling may be impractical
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