Dataset DOI: 10.5061/dryad.xsj3tx9rf

Description of the data and file structure

This dataset contains all routines and raw data for the manuscript 'Quantifying 3D Live-Cell Membrane Dynamics Using Dynamic Metal-Induced Energy Transfer Spectroscopy (dynaMIET)'. 

Readme for the dataset

Explanation of the original data file

All the data are collected with SymphoTime software (v. 2.8) by PicoQuant GmbH, Berlin (software accompanying their MicroTime single-molecule spectroscopy systems: https://www.picoquant.com/products/category/software/symphotime-64-fluorescence-lifetime-imaging-and-correlation-software).

Explanation of the data plotting method

All the raw data for plotting the figures are in the 3DMembraneDynamics_Data&Plot.

Plot-figures were drawn by Mathemaica 13.2. The detailed code is list in the 3DdynamicPlotFigureTao.nb.

Files and variables

File: GUV_point.ptu/GUV_400mOsmL_30degree.ptu/GUV_450mOsmL.ptu

Description: original data file for the giant unilamellar vesicles membrane point measurements on different conditions.

File: SLB_point.ptu

Description: original data file for the supported lipid bilayer measurement;

File: Cell_PM_point.ptu

Description: original data file for the cell plasma membrane point measurement;

File: Cell_ER_point.ptu

Description: original data file for the endoplasmic reticulum point measurement;

File: Cell_ER_image.ptu

Description: original data file for the endoplasmic reticulum imaging measurement;

File: Cell_PM_image.ptu

Description: original data file for the cell plasma membrane imaging measurement;

File: Cell_NE_image.ptu

Description: original data file for the nuclear envelope membrane imaging measurements;

File: Cell_NE_point.ptu

Description: original data file for the nuclear envelope membrane point measurements;

File: GUV_image.ptu

Description: original data files for the giant unilamellar vesicles membrane imaging measurements;

File: 3DMembraneDynamics_Data_Plot.zip

Description: raw plotting data

Code/software

*dynaMIET analysis*

File: dynaMIET_MatlabCode.zip

Description: Thus folder contains all the codes for analyzing the dynamics and images from the .ptu file. The main routine for point measurement is Main_3DMembraneDynamics.m and for scanning measurment is Main_Membrane_height.m. These scripts depend on additional m-files provided within this repository. File xx.mexw64 is a compiled MEX (MATLAB Executable) binary for 64-bit Windows, designed to be used within MATLAB. It is not a standalone file and cannot be opened directly. Instead, it is automatically executed within MATLAB as part of the analysis workflow when running the provided scripts.

Method to extract data from .ptu file:

For the following point measurement files:

GUV_point.ptu, GUV_400mOsmL_30degree.ptu, GUV_450mOsmL.ptu,

SLB_point.ptu, Cell_PM_point.ptu, Cell_ER_point.ptu, Cell_NE_point.ptu

Steps:

Copy the folder path (important: not the full file path ending with .ptu).

The routine will automatically detect the .ptu file within the folder.

Paste the folder path into:

Line 20 and Line 44 of Main_3DMembraneDynamics.m

Run the script:

Evaluate Section 1 (Lines 1–120)

If fitting is required:

Paste the same folder path into Line 124

Evaluate Section 2 (Lines 124–154)

For the following scanning measurement files:

Cell_ER_image.ptu, Cell_PM_image.ptu, Cell_NE_image.ptu, GUV_image.ptu

Steps:

Copy the folder path (important: not the full file path ending with .ptu).

Paste the folder path into:

Line 12 of Main_Membrane_height.m

Run the entire script.

3D dynamics simulation

File: membrane_3D_dynamics_simulation.zip

Description: This folder contains all the codes for 3D membrane simulation (Figure 1D and Figure 1E), the main routine is the Main_Mmbrane_3Dynamics.m.

Figure 1D: Simulation of a fluctuating membrane above a gold surface, showing the corresponding fluorescence intensity and lifetime traces. These traces illustrate how both the fluorescence intensity and lifetime of the labeled membrane vary as a function of its height.

Figure 1E: Calculated height correlation curve derived from the simulation, showing the characteristic timescale and amplitude of membrane height fluctuations.

*Calculation of brightness isosurfaces*

File: MIET_calc-main2.zip 

This folder contains the Python code used to calculate molecular brightness isosurfaces. The main entry point is MIET_3Disosurface.py. In addition, the MATLAB script IsosurfaceCal.m provides an alternative implementation for calculating the molecular brightness isosurface ( Figure 1C).

Figure 1C: The comparison of calculated iso-surfaces of molecular brightness as a function of position within a confocal focus (Numerical aperture = 1.49) of a circular polarized beam, with the focal plane positioned at the glass–sample interface, for both a glass substrate and a gold substrate. 

More details can be found at GitLab/GitHub (https://gitlab.gwdg.de/tchen1/3dmembranedynamics) and (https://github.com/Nkaredla/MIET_calc). The original code was developed by a co-author of this study, who has agreed to its release under CC0 license for the purpose of this publication.

Data analysis workflow

1) Calculate the lifetime and brightness calibration curves

The Matlab program that calculates the theoretical curves for Figure 1B is CalibrationCal.m or MIETcurvefunction.m. It needs some the m-files contained in this repository. A PDF file (LifetimeL.pdf) describs the routine LifetimeL.m used by MIETcurvefunction.m. The MIETcurvefunction.m also can be directly called in Main_3DMembraneDynamic.m. The optical parameters of dye and substrate are needed for the calculation. 

Figure 1B: Calculated relative fluorescence lifetime and emission brightness of an emitter as a function of its distance from a gold-modified substrate. The curves illustrate how near-field interactions with the metal surface modulate the emitter’s radiative and non-radiative decay rates, leading to distance-dependent changes in both fluorescence lifetime and intensity.

2) Calculate the fluorescence intensity trace and lifetime trace (Figure 2D)

The routine plotlifetime_trace_fit.m is used to calculate the fluorescence intensity trace and lifetime trace from a ptu file. The time bin width is needed for the calculation. The plotlifetime_trace_fit.m also can be called directly from Main_3DMembraneDynamic.m. The result can be saved automatically.

3) Calculate the height trace (Figure 2D)

Main_3DMembraneDynamic.m contains code directly converts the lifetime trace to height trace based on the MIET calibration curve.

Figure 2D: Calculated fluorescence intensity time trace, height trace, and brightness trace for the proximal membrane of one deflated giant unilamellar vesicles. 

4) Calculate the intensity autocorrelation function and height autocorrelation function (Figure 2E, 2F, 2H, 2I, Figure 3C, 3E，Figure 4C, 4E, Figure 5C, 5E)

The intensity autocorrelation function and height autocorrelation function can be calculated in the Main_3DMembraneDynamic.m. through routine tttr2xfcs.m. Check fluorescence photobleaching correction is appled or not.

Figures 2E, 2H, 3C, 4C, and 5C: Intensity autocorrelation functions measured for giant unilamellar vesicles (Figure 2E), supported lipid bilayers (Figure 2H), the plasma membrane (Figure 3C), the endoplasmic reticulum (Figure 4C), and the nuclear envelope (Figure 5C).

Figures 2F, 2I, 3E, 4E, and 5E: Height autocorrelation functions corresponding to giant unilamellar vesicles (Figure 2F), supported lipid bilayers (Figure 2I), the plasma membrane (Figure 3E), the endoplasmic reticulum (Figure 4E), and the nuclear envelope (Figure 5E).

5) Fit the intensity autocorrelation curve (Figure 2E, Figure 2H, Figure 4C, Figure 4C, Figure 5C)

The routine Dynamics3D_tri.m can be called in Main_3DMembraneDynamic.m to fit the intensity autocorrelation curve. The diffusion coefficient is obtained directly. The fitting mode contains the tri-state equation, if it's not applicable, using one parameter in the value of estimated diffusion time, the routine will automatically omit the tri-state for fitting.

6) Fit the height autocorrelation curve (Figure 2F)

The routine HeightCorrFit.m can be called in Main_3DMembraneDynamic.m to fit the height autocorrelation curve. Note that for different membranes, parameters in HeightCorrFit.m must be changed accordingly.

7) View data & save

 3D height profiles

All the 3D height profiles were created by a open-source sofeware MIET-GUI (Ghosh, A., Chizhik, A.I., Karedla, N. et al. Graphene- and metal-induced energy transfer for single-molecule imaging and live-cell nanoscopy with (sub)-nanometer axial resolution. Nat Protoc 16, 3695–3715 (2021). https://doi.org/10.1038/s41596-021-00558-6).