A Multifunctional Probe for Visualization of the Nanoscale Distribution of Cholesterol in Cells by Expansion Microscopy

Abstract

Unraveling the nanoscale distribution of small molecules in cells is of central importance for the understanding of cellular functions and the development of drugs. However, particularly the visualization of lipids such as cholesterol—a central compound of cell membranes—with high spatial resolution remains challenging because they cannot be efficiently immobilized for super-resolution microscopy investigations. Here we developed an azido- and amino-modified cholesterol probe that can be efficiently fixed and labeled with fluorophores by click chemistry. In combination with expansion microscopy, its cellular localization and interaction with other cellular proteins can be precisely determined in fixed cells at varying time points after addition. Our approach allows us to detect the endocytic pathway of cholesterol with unprecedented spatial resolution and shows that cholesterol is efficiently ingested in endocytic vesicles and accumulates as cholesterol aggregates with an average size of ∼37 nm in late endosomes and lysosomes, respectively.

Keywords

expansion microscopy / multifunctional cholesterol probes / super-resolution / tracking

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2025 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.