Supporting Dataset for Publication: Refractive index mapping below the diffraction limit via single molecule localization microscopy

Description of dataset

This dataset includes all necessary information to reproduce the analysis and figures from the publication " Refractive index mapping below the diffraction limit via single molecule localization microscopy". It includes all figures from the main manuscript (Fig.1-Fig.3), as well as the supporting figures (Fig S1-S6) in high resolution. Furthermore, the dataset includes a detailed description of the used software, packages, python scripts and online repositories. Additionally, the dataset entails the localisation data files.

Authors

Simon Jaritz¹, Lukas Velas¹, Anna Gaugutz¹, Manuel Rufin², Philipp J. Thurner², Orestis G. Andriotis², Julian G. Maloberti³, Simon Moser³, Alexander Jesacher³, Gerhard J. Schütz^{1, ‡}

⁽¹⁾ Institute of Applied Physics, TU Wien, Vienna, Austria
⁽²⁾ Institute of Lightweight Design and Structural Biomechanics, TU Wien, Vienna, Austria
⁽³⁾ Institute of Biomedical Physics, Medical University of Innsbruck, Müllerstraße 44, 6020 Innsbruck, Austria

^‡ Contact Person details: schuetz@iap.tuwien.ac.at 

Technical details

Reproduction of the figures: 
High Res Images: Features all Figures (Figures 1-3 and Supplementary Figures S1-S6) from the Paper, in high resolution (600 dpi).

• Fig 1a: was created using power point
• Fig 1b and c: all necessary scripts and data to create Fig 1b and Fig 1c are presented in the folder “Fig1b_and_Fig1c PSF.zip”
• Fig 1d:  was created using the script in the folder “theoretical RI measurement.zip”
• Fig 2: all necessary data to create Fig 2 are presented in the data folder and in the Online Repository: “SMLM-Analysis” by Simon Jaritz. The script to create Fig2 is called: “comparing_results.ipynb“
• Fig 3: all necessary scripts and data to create Fig3 are presented in the data folder and in the Online Repository: “SMLM-Analysis” by Simon Jaritz. The script to create Fig3 is called: “profile change over length of fibril.zip“

• Fig S1: was created using the script: “theoretical RI measurement.zip”
• Fig S2: was created using the script: “Print_STORM_AFM_Profile.ipynb.zip”
• Fig S3: all necessary scripts and data to create Fig 1b and Fig 1c are presented in the folder “Change along the fibril FigS3.zip”
• Fig S4: was created using the script in the folder “theoretical RI measurement.zip”
• Fig S5: was created with a free version of AutoCAD web
• Fig S6: was created using the data and scripts in the file “fibril analysis.zip”

Required Software 

• Matlab (Mathworks), version R2023b
• Python version 3.11.5, for determining the single molecule localizations, in combination with the Matlab software provided by A.Jesacher and J.Maloberti
• Python (version 3.9.13), for performing the analysis and create the figures in the paper

All Python software requirements and packages, are listed in the file python_requirements.txt

Analysis Scrips and Files-Folder

• File: aberration measurement.zip (from Alexander Jesacher et.al):
• Matlab Application to calculate the aberrations of the optical setup
  for single molecule analysis
• File: aberrations.mat
  Used aberration file for the single molecule analysis, calculated using the Matlab app aberration measurement (see below)
• File: theoretical RI measurement.zip (from Alexander Jesacher et.al):
• Python program for the theoretical precision determination of the refractive index, used for Fig.1d, FigS1 and FigS4
  
  

Online Repositories

• Online Repository: mlefitgpu (from Julian Maloberti)
  • Python program to determine the fitted PSF as well as performing the GPU fit,
    to determine the 3D position of recorded microscopy data (SMLM localisations)
  • available on: https://github.com/jgmaloberti/mlefitgpu
    acces to that code is restricted and is managed by Julian Maloberti
• Online Repository: ForceMapAnalysis (from Manuel Rufin)
  • Matlab program to analyze and transform AFM data
  • available on: https://github.com/Rufman91/ForceMapAnalysis
• Online Repository: SMLM-Analysis (from Simon Jaritz)
  • Collection of Python scripts that were used to make the fibril analysis
  • available on: https://github.com/simonjaritz/SMLM-Analysis

Localization Data files

The localization data-folder contains the necessary Data (point clouds) to perform the fibril analysis.
Please note that the RAW data (tiff and jpk-files) recorded with the microscopes, is only available upon request.

 All region of interests (ROI) contain the same file strucutre and below is an example of ROI1:

• ROI 1:
  • AFM_pointcloud_...-dry.csv: AFM dry data, stored as point clouds
  • AFM_pointcloud_...-Qi-liquid-fit.csv: AFM wet data stored as point clouds
  • Fibril_cutouts.pkl: cutout coordinates for the fibrils
  • List_of_val.csv: defocus value and middle layer thickness for each fibril
  • Overlay_table.pkl: transformation parameters for the overlay
  • ROI 1 overview.png: shows which fibril is which in an overview image
  • ROI 1 SMLM files folder:
    • Single molecule Localization files, drift corrected
      These files contain the localizations that were analyzed using the Script mlefitgpu from Julian Maloberti (see below), with the following code example: “results_def_579_mid_138_n133_n1.4_n1518.csv”
      
      objective defocus = 579 nm
      middle layer thickness (AFM fibril height) = 138 nm
      refractive index surrounding medium = 1.33
      refractive index collagen = 1.40
      refractive index glass coverslip = 1.518