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

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: 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: 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: 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