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Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy
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Refractive Index Mapping below the Diffraction Limit via Single Molecule Localization Microscopy.

Simon Jaritz1, Lukas Velas1, Anna Gaugutz1

  • 1Institute of Applied Physics, TU Wien, 1060 Vienna, Austria.

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|December 26, 2025
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Summary
This summary is machine-generated.

Combining single molecule localization microscopy (SMLM) and atomic force microscopy (AFM) enables subdiffraction mapping of refractive index in biological samples. This reveals structural heterogeneity in single collagen fibrils, offering insights into their hydration and molecular organization.

Keywords:
3-dimensional single molecule localization microscopyatomic force microscopycollagen fibrilsdSTORMdefocused imagingmultimodal imagingrefractive index

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Area of Science:

  • Biophysics
  • Microscopy
  • Materials Science

Background:

  • Single molecule localization microscopy (SMLM) images biological samples in 3D below the diffraction limit.
  • The point spread function's shape in SMLM offers data on sample refractive properties.
  • Atomic force microscopy (AFM) provides high-resolution surface topography.

Purpose of the Study:

  • To develop and demonstrate a method combining SMLM and AFM for subdiffraction refractive index mapping.
  • To investigate the refractive index and hydration of individual collagen fibrils.
  • To reveal nanoscale structural heterogeneity within collagen fibrils.

Main Methods:

  • Integration of SMLM and AFM for correlative imaging.
  • Utilizing SMLM's point spread function analysis to determine refractive index.
  • Applying AFM for precise topographical measurements of biological samples.

Main Results:

  • Successful mapping of refractive index in biological samples at subdiffraction resolution.
  • Determination of refractive index variations and swelling behavior in single collagen fibrils.
  • Identification of nanoscale fluctuations in refractive index along collagen fibrils, indicating structural heterogeneity.

Conclusions:

  • The combined SMLM-AFM approach precisely maps refractive index at the nanoscale.
  • Single collagen fibrils exhibit heterogeneous refractive indices and differential hydration.
  • This method uncovers structural variations within collagen fibrils at the molecular level.