Work

Refractive Index Mapping below the Diffraction Limit via Single Molecule Localization Microscopy

Year: 2025

Type: article

Abstract: Single molecule localization microscopy (SMLM) is a powerful method to image biological samples in three dimensions, below the diffraction limit of light microscopy. Beyond the position of the emitter, the shape of the single-molecule point spread function provides additional information, for example, about the refractive properties of the sample between the emitter and the glass coverslip. Here, we show that the combination of SMLM with atomic force microscopy (AFM) allows mapping of the refractive index of a biological sample at subdiffraction resolution and at a precision only limited by measurement errors of SMLM and AFM. We showcase the method by the determination of the refractive index of isolated single collagen fibrils. Variabilities both in refractive index and the swelling behavior of single fibrils upon drying and rehydration exposed deviations from the ensemble behavior, demonstrating differential hydration of single collagen fibrils. Mapping the refractive index along single collagen fibrils revealed substantial fluctuations at characteristic length scales below 500 nm, which indicates the structural heterogeneity of collagen fibrils at the length scale of single collagen molecules.

Source: ACS Nano

DOI: 10.1021/acsnano.5c17647