Work

Maximising imaging volumes of expanded tissues for inverted fluorescence microscopy

Year: 2025

Type: article

Abstract: Expansion microscopy (ExM) has enabled nanoscale imaging of tissues by physically enlarging biological samples in a swellable hydrogel. However, the increased sample size and water-based environment pose challenges for deep imaging using conventional inverted confocal microscopes, particularly due to the limited working distance of high-numerical-aperture (NA) water immersion objectives. Here, we introduce a practical imaging alternative that utilizes an inverted water-dipping objective and a refractive-index-matched optical path using fluorinated ethylene propylene (FEP) film. Through point spread function (PSF) measurements and simulations, we show that the FEP film introduces predominantly defocus-like wavefront profiles characteristic of high NA systems, which result in an easily correctable axial shift of the focal plane. To ensure stable immersion and refractive index continuity, we use an arrangement relying on an FEP film, Immersol W, water and a FEP-based imaging dish. This configuration achieves sub-micron lateral and axial resolution, supports large tile-scan acquisitions, and maintains image quality across depths exceeding 800 µm. We validate the system by imaging 4×-expanded U2OS cells and human cerebral organoids. Our approach provides a low-cost, plug-and-play solution for high-resolution volumetric imaging of expanded samples using standard inverted microscopes.

Source: Biomedical Optics Express

DOI: 10.1364/boe.579043

Authors: Miguel Cardoso Mestre, Jacob R. Lamb, Madeline A. Lancaster, James D. Manton

Institution: MRC Laboratory of Molecular Biology

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,913

Funder: Royal Society