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

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.