DOI: 10.48436/shmmj-7w030
title: Supporting Dataset for Publication: Refractive index mapping below the diffraction limit via single molecule localization microscopy

description: 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. Schütz1, ‡

 

(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/mlefitgpuacces 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 correctedThese 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 nmmiddle layer thickness (AFM fibril height) = 138 nmrefractive index surrounding medium = 1.33refractive index collagen = 1.40refractive index glass coverslip = 1.518






 
description: 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 combination of SMLM with atomic force microscopy (AFM) allows to map the refractive index of a biological sample at sub-diffraction resolution and at a precision only limited by measurement errors of SMLM and AFM. We showcase the new 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 structural heterogeneity of collagen fibrils at the length scale of single collagen molecules.