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

## TL;DR

This paper introduces a new method to map refractive index at subdiffraction resolution using SMLM and AFM, revealing structural details in collagen fibrils.

## Contribution

Combining SMLM with AFM to map refractive index at subdiffraction resolution, revealing structural heterogeneity in collagen fibrils.

## Key findings

- Refractive index of single collagen fibrils was mapped with subdiffraction resolution.
- Collagen fibrils showed structural heterogeneity at scales below 500 nm.
- Swelling behavior and refractive index varied between individual fibrils.

## 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.