Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

In our research we investigate the influence of the mechanical properties of the extracellular matrix on cell behavior and tissue integrity. We want to understand how the interplay between cells and their microenvironment affects pathological processes like metastasis formation. By data mining the mechanical properties of the pulmonary basement membrane we discovered that softer basement membranes are more resistant to cancer cell invasion.

The method presented in our protocol enables the cryosectioning and mechanical investigation of unfixed biological samples, which preserves the mechanical properties. And furthermore, it shows a way to determine the Young's modulus solely of the basement membrane inside the alveolar wall by an atomic force microscope measurement and filtering procedures described in our protocol. The next step is to learn more about the dynamics of basement membrane stiffness and its role in developmental and pathological processes to potentially improve diagnostics and guide treatment options.

To begin, remove the mold containing the frozen optimal cutting temperature, or OCT, compound embedded mouse lung tissues from the minus 80 degrees Celsius freezer. Place double-sided adhesive tape at the center of the frosted edge microscope slide. Roll a 15-milliliter centrifuge tube over the tape to ensure firm and bubble-free adhesion.

Ensure the length of the tape matches the width of the OCT block containing the sample. Label the slides with a pencil, including the sample information and section numbers. Insert the prepared microscope slides into a slide box.

Place the slide box inside the cryotome chamber to chill the slides. Next, fill the inner two rings of the sample holder with OCT medium. Position the sample within the OCT medium on the sample holder.

Place the sample holder in the cryotome chamber for approximately 10 minutes until the OCT medium solidifies completely and the sample is firmly secured. Now, install the sample holder onto the cutting stage of the cryotome. Attach a piece of one-sided adhesive tape to the sample by pressing firmly with a cool thumb.

Produce a 15-micrometer tissue section using the cryotome. Employ a brush to direct the section and prevent the adhesive tape from detaching during the cutting procedure. Use a chilled microscope slide with double-sided adhesive tape and press it firmly onto the section to pick it up.

Place the slide carrying the section back into the slide box. To calibrate the cantilever using the thermal noise method, place a microscope slide on the sample stage of the atomic force microscope, or AFM. Place the AFM head on the sample stage.

Then lower the AFM head using the stepper motors until the gap between the cantilever holder and the microscope slide is approximately one to two millimeters. Using a one-milliliter syringe with a long needle apply PBS to the side of the cantilever holder, allowing it to flow down and form a fluid meniscus in the gap. In the AFM control software to perform a contact-based calibration navigate to the Acquire Data page and select the Advanced View option.

Access the Calibration Manager by clicking the burger menu button located in the top-right corner. Then select Contact-based as the method, and MLCT-F cantilever from the Name dropdown menu as the cantilever name. Enter one volt in the Setpoint field, and 10 in the Number of scans field.

On the Acquire Data page input a Setpoint of one volt for the automatic approach procedure in the left-hand control panel. Click the blue downwards pointing arrow button in the top-left corner of the interface to initiate the automatic approach. When the approach is complete and the cantilever is in contact with the microscope slide, click the Calibrate button in the Calibration Manager window to start calibration.

After calibration is complete close the Calibration Manager window and ensure that a calibration file is generated in the pre-selected directory containing the determined calibration results. To begin, retrieve the specimen slide containing sectioned mouse lung tissues from the freezer. Place the microscope slide on the sample stage of the AFM.

Click on the button with the wrench icon in the upper-right corner of the user interface to navigate to the Settings Manager. Within the Approach Settings section set the Target Height to four micrometers. Then in the Current Mode Settings section under Advanced Feedback Settings, set the multiplier to one.

In the Force Settings subsection of the Current Mode Settings select Retracted Piezo from the Mode at end dropdown menu. In the left-hand control panel set the parameters for the force indentation curves. For the NanoWizard 4 XP and the MLCT cantilever F input Setpoint to five nanonewtons, Z Length to eight micrometers, and Z Velocity to 300 micrometers per second.

Next, define the location and size of the initial overview force map to encompass the entire alveolar wall, extending from one air side to the other. Set the number of pixels to 50 by 50. After recording the overview map, capture a more focused force map with a size of either three by three micrometers or four by four micrometers on the basement membrane, keeping the number of pixels at 50 by 50 curves.

To analyze force indentation curves launch the CANTER Processing Toolbox in MATLAB and open the Force Curve Analysis application. Then to load the high resolution force map of the lung section click Select File, navigate to the save location, double-click on the file, and select Load Data. When the pop-up window requesting calibration values for the cantilever appears, enter the calibration values in the respective edit fields.

Click the Submit button to proceed. Upon a second pop-up window continue with the loading procedure of the quantitative imaging, or QI, map by clicking the Submit button. Upon completion of the loading process the initial force curve of the force map is displayed on the screen.

Set the fit depth in the corresponding edit field to 1.5 micrometers and select via Hertz fit for the contact finder algorithm. To apply the fit of the modified Hertz model to all force indentation curves of the QI map, click the Keep Apply to all button. After completion of the last force curve analysis a window appears to save the files.

Click Yes and enter a name for the result files. For spatial filtering of QI map from the Application Selection window in the CANTER Processing Toolbox, select the Result Filtering Tool and click on the Start Application button. To load the fit results click on Open in the top menu bar of the Result Filtering Tool user interface.

In the subsequent popup window locate the first Set button under the JPK Maps tab and select the tsv file containing the force curve analysis outcomes. Then click on the second Set button and locate the QI map file corresponding to the selected tsv file. Subsequently, click Submit to load the map data and force curve analysis results.

From the Displayed channel dropdown menu at the top select the EModul option to display Young's modulus results as a map image. Then from the Data channel dropdown menu below the histogram plot choose the EModul option to visualize the distribution of the loaded Young's modulus values of the QI map. Next, click on the Manipulation flow arrow button in the center of the user interface, ensuring it points towards the right-hand side.

Set the Image filter toggle button to the On position in the Filter panel above the histogram axes. From the Filter geometry dropdown menu select the Freehand option and then click on the Add button. On the top channel image draw the filter mask by circling the basement membrane while holding the left mouse button.

Then release the mouse button when finished and double-click on the mask to apply it to the map. To save a new tsv result file with the masked Young's modulus values click on Save, Save histogram, and Save data in the top menu bar. In the popup window that appears click Select all to choose which results to write to the tsv file.

Then click on the OK button to confirm the selection. In the Save dialog enter a name for the tsv file, choose the desired save location, and click OK to save the filtered results. The Young's modulus values of the pulmonary basement membrane exhibited a log normal distribution with a peak value of 9.31 and standard deviation of 0.18, yielding a representative Young's modulus of 11.05 kilopascals.

These findings were further confirmed by quantile-quantile plot.