DOI: 10.5061/dryad.v41ns1s60
title: A look beyond topography: transient phenomena of Escherichia coli cell division captured with high-speed in-line force mapping

description: Life on the nanoscale has been made accessible in recent decades by the
 development of fast and non-invasive techniques. High-speed atomic force
 microscopy (HS-AFM) is one such technique that sheds light on single
 protein dynamics. Extending HS-AFM to effortlessly incorporate mechanical
 property mapping while maintaining fast imaging speed allows us to look
 deeper than topography and reveal details of the nanoscale mechanisms that
 govern life. Here, we present high-speed in-line force mapping (HS-iFM),
 which enables the recording of mechanical properties and topography maps
 with high spatiotemporal resolution. Employing HS-iFM, a comprehensive
 study of the nanoscale mechanical properties of
 living Escherichia coli revealed localized stiffening and details
 during cell division, formation and diffusion of pores in the membrane,
 and the impact of depressurization of a cell. The frame time was as low as
 15 seconds with a spatial resolution of 5.5 nm/pixel in topography and 22
 nm/pixel in force maps, allowing the capture of transient phenomena on
 bacterial surfaces in striking detail.
description: # A look beyond topography: transient phenomena of *Escherichia coli* cell
 division captured with high-speed in-line force mapping
 [https://doi.org/10.5061/dryad.v41ns1s60](https://doi.org/10.5061/dryad.v41ns1s60) ## Description of the data and file structure Data was collected with high-speed atomic force microscopy (HS-AFM), and force maps were created using high-speed inline force mapping (HS-iFM). Optical/fluorescence microscopy data was collected with an optical microscope using a 100x objective lens. ### Files and variables #### File: ads3010\_SciAdv\_Ganser\_et\_al.zip **Description:** All raw data for the publication "A look beyond topography: transient phenomena of *Escherichia coli* cell division captured with high-speed in-line force mapping" are included in folders named after the figures in the article. There are specific "overview"-files contained in each folder describing the contents. The file structure is as follows: **Folder "fig2_fig3_figS7_figS8_figS9"**: Contains HS-AFM topography, HS-iFM force map, and fluorescence microscopy measurements of an *E. coli* cell dividing. HS-AFM topography files (ASD-files) are successively recorded and stacked 2D maps of height values and can be opened with NanoLocz (see "Software" section), fluorescence microscopy files (CXD) are video recordings of a CCD camera and can be opened with Fiji (see "Software" section). HS-iFM force maps (DFM) contain the raw force curve data for each pixel and each frame. The code to open these DFM files is provided in the "Software" folder. DFM files were analyzed to extract elastic modulus maps, which are provided as IBW, ASD, and TIFF files. IBW requires Wavemetrics IgorPro, while ASD can be opened by NanoLocz as mentioned above, and TIFF files can be opened by most image viewers. In the following, a detailed description of which files contain the data described above is given and how the files can be identified according to their naming conventions: * subfolder "fluorescence_microscopy" or "optical_microscopy": contains fluorescence microscopy or bright field microscopy data of the dividing bacteria (CXD files) * subfolder "HS_iFM": contains raw and analyzed HS-iFM data * sub-subfolder "analyzed": Modified data; naming convention: DATE_NUMBER_DESCRIPTOR.EXTENSION * DATE: Date of recording * NUMBER: Sequential number assigned during recording. If NUMBER="combined", it indicates a file combined from all individual files * DESCRIPTOR: * \[Nothing]: Height map (AFM topography) * Modulus: Elastic modulus map (units: Pa) * Shifted: drift corrected height map * Shifted_Filtered: Drift-corrected and high-pass filtered topography map * Modulus_Shifted: Drift-corrected elastic modulus Map * EXTENSION: * ASD: can be opened by open source program NanoLocz (see below) * IBW: can be opened by IgorPro (commercial software) * Always ASD and IBW are provided together * sub-sub-subfolder: modulus_combined_shifted * contains the analyzed elastic modulus data as TIFF files, combined and drift-corrected * sub-subfolder "raw": Raw data of the recording, naming convention: DATE_NUMBER.EXTENSION * DATE: Date of recording * NUMBER: Sequential number assigned during recording. * EXTENSION: File extension * ASD: HS-iFM topography data * DFM: Force map data containing individual force vs. distance curves. The C++ code to read DFM files is provided in the folder "software/force_map_loading" * subfolder "plots": Contains data extracted from HS-iFM data to be used for plotting graphs. * sub-subfolder "lineprofiles": * AutoLineTimeSeries_NUMBER: elastic modulus line profile along the bacteria for each frame recorded * AutoTopoLineTimeSeries_NUMBER: height line profile along the bacteria for each frame recorded, corresponding to the elastic modulus line profile with the same number * AutoTopoLineTimeSeries_perpendicular: Line profile perpendicular to the other line profiles, used to estimate the bacteria cell radius * sub-subfolder "results": results of the contact mechanical analysis and average elastic modulus determination, according to the scripts provided in "software/contact_mech_analysis_and_e_modulus_plots" * one or more files "NAME.csv" or "NAME.txt": contain the thermal spectrum of the HS-AFM cantilever used; for spring constant calibration * "overview.txt": a brief overview of the files and the results of the cantilever calibration **Folder "fig4_figS12_figS13_figS16"**: Contains HS-AFM topography, HS-iFM force map, and bright field microscopy measurements of an *E. coli* cell dividing at high resolution. The folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". * Additional to the previously described folder structure, there are files in "fig4_figS12_figS13_figS16/HS_iFM/analyzed" containing "C1" or "C2" and "mag". "C1"/"C2" describes clipped portions of a file, while "mag" indicates magnification. * 20230928_020017_C1.asd: HS-iFM topography; clipped portion of "20230928_020017.asd" to show the *E. coli* division site * 20230928_020017_C1_mag.asd: HS-iFM topography; Magnified division site of "20230928_020017_C1.asd" * 20230928_020017_C2.asd: HS-iFM topography; further clipped to show bridges rupturing during cell division * 20230928_020017_C2_mag.asd: HS-iFM topography; magnified and further clipped to show bridges rupturing during cell division * The folder "pores" in "fig4_figS12_figS13_figS16/HS_iFM/analyzed" contains four files visualizing pores on the *E. coli* surface * tt1.asd: Drift corrected portion of "raw/20230928_020016.asd" * tt1_mag.asd: Magnified region of tt1.asd * tt1_mag_shifted.asd: Further drift corrected and high-pass filtered (from tt1_mag.asd) * tt1_mag_shifted_mag.asd: Further magnification of tt1_mag_shifted.asd, to clearly visualize nanometer scale pores **Folder "fig5_figS14":** Contains HS-AFM topography, HS-iFM force map, and bright field microscopy measurements of an *E. coli* cell bursting during observation. The folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". **Folder "figS1":** Contains files used to plot signal traces during HS-iFM measurements. * osci_traces.txt: Full recording of deflection, amplitude, controller trigger (PID hold) signal, and z-piezo voltage during HS-iFM measurements to demonstrate the methodology. All units and scales are presented in the "plot_*.plt" files. * plot_raw_force.plt: gnuplot file to plot "raw_force.txt" * plot_traces.plt: plots 12 ms of all channels in "osci_traces.txt" * plot_traces_zoom1.plt: plots a few cantilever oscillation cycles between force applications from "osci_traces.txt" * plot_traces_zoom2.plt: plots a force application from "osci_traces.txt" * raw_force.txt: raw force vs. distance curve as recorded by HS-iFM **Folder "figS2":** Contains files used to plot the dependency of the HS-iFM frame time to several variables and determine which variables influence the frame time in HS-iFM in terms of generating an overhead time. * fit_results.txt: Plane fit of overhead time vs. topography time and force curve time * force_curve_points.txt: Overhead time vs. points in a single force curve * force_map_pixel.txt: Overhead time vs. pixel number in the force map * force_time1.txt: Overhead time vs. individual force curve time at 1s topography time * force_time10.txt: Overhead time vs. individual force curve time at 10s topography time * frame_force_time_plot.plt: Plots 3D plot of fit_results.txt, frame_time.txt, force_time1.txt, and force_time10.txt using gnuplot * frame_time.txt: Overhead time vs. force curve time and topography time * plot_curve.plt: Plots overhead time vs. topography pixel number (can be used to plot any of the individual 2D plots) using gnuplot * topo_pixel.txt: Overhead time vs. topography pixel number **Folder "figS3":** Contains data to show the principle of removal of stripes caused by force measurements in HS-iFM. * plot_profile.plt: Plots both of the files contained in this folder using gnuplot. * profile_corr.txt: Shows the corrected line profile, where trenches are removed by linear interpolation of adjacent points * profile_uncorr.txt: Shows a topography line profile on an *E. coli* cell distorted by deep trenches (caused by HS-iFM force mapping) **Folder "figS4":** Contains HS-AFM topography and HS-iFM force map data measured on PDMS substrates to verify that HS-iFM can produce quantitative results. While slightly simpler, the folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". **Folder "figS10"**: Contains HS-AFM topography, HS-iFM force map, and bright field microscopy measurements of an *E. coli* cell dividing. The folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". **Folder "figS11"**: Contains HS-AFM topography, HS-iFM force map, and bright field microscopy measurements of an *E. coli* cell dividing. The folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". **Folder "figS15":** Contains HS-AFM topography, HS-iFM force map, and bright field microscopy measurements of an *E. coli* cell bursting during observation. The folder structure, naming conventions, etc. are the same as in the folder "fig2_fig3_figS7_figS8_figS9". **Folder "software":** Contains code used for the analysis of the data. * **contact_mech_analysis_and_e_modulus_plots:** Used to analyze data found in the "lineprofiles" folders, as described above. Follow the guide below, on how to perform the analysis: * GNU/Octave (tested with version 9.2.0 * -requires "optim" package * (1) average_radius_development("path_to_lineprofiles", NumberOfProfiles) * Monitor the individual fit results and adjust fit range and minimum initial value estimation as necessary. Note that the results radius values before the division site is notably developed will scatter wildly and are extremely sensitive to to the initial parameters. After the division site has developed the scattering and sensitivity are much improved. * Example: * ``` average_radius_development("../../figS10/plots/20230925_020010/AutoTopoLineTimeSeries", 9) ``` * Results: R2l_min.txt * (2) estimate_cell_radius("path_to_file", NrToPlot) * Check with the plot and the results and adjust parameters as needed * Example: * ``` estimate_cell_radius("../../figS10/plots/20230925_020010/AutoTopoLineTimeSeries_perpendicular.txt", 10) ``` * Results: R2p.txt * (3) auto_correction([R1x,R1y],dilationFlag) * Hertzian correction of local curvature. * \[R1x,R1y] ... tip radius if approximately spherical R1x = R1y * dilationFlag ... 0 -&gt; no dilation correction / 1 -&gt; dilation correction * Example: * ``` auto_correction([5,5],1) ``` * Results: correction_factor.txt * (4) average_stiffness_change(folder, topo_basename, mod_basename, N) * Calculates the average elastic modulus of a cell and the relative increase of the elastic modulus of the division site (stiffening_ratio.txt) * Example: * ``` average_stiffness_change("../../figS10/plots/20230925_020010", "AutoTopoLineTimeSeries", "AutoLineTimeSeries", 9) ``` * Results: stiffening_ratio.txt, elastic_modulus.txt - **force_map_analysis**: This code is used to analyze DFM files after loading them with the code in "force_map_loading". The code was written in Wavemetrics IgorPro 9. For use with other software or as a standalone script, it will need to be adapted. - **force_map_loading**: This code is written in C++ and is used to load DFM files into the memory for further analysis with the code in "force_map_analysis". This code needs to work in concert with Wavemetrics IgorPro 9 and needs Wavemetrics development resources in order to be compiled as is. However, the function *extern "C" int d_load_force_map()* describes the loading process of DFM files and can be used to adapt the software for standalone use. ## Code/software The code to read DFM files (containing force map data) is included in the "software" folder. IBW files (e.g. elastic modulus maps) can be opened with IgorPro (saved and verified with version 9) ASD (e.g. HS-AFM topography, elastic modulus map) files can be read with e.g., the open source NanoLocz ([https://github.com/George-R-Heath/NanoLocz](https://github.com/George-R-Heath/NanoLocz)) CXD files can be opened with Fiji ([https://fiji.sc](https://fiji.sc)) PLT files can be plotted with Gnuplot ([http://www.gnuplot.info](http://www.gnuplot.info)) ## Access information N/A