This protocol shows a step-by-step guide to measure the stiffness of hydrogels and cells using a commercially available nanoindenter and also presents an open-source software to reproducibly analyze acquired data. The protocol allows us to obtain atomic force microscopy-like data, however, at a fraction of the complexity. So this protocol will be useful for scientists interested in studying mechanical properties of healthy and diseased samples, but also we believe it's going to be of broader applicability in the context of nanoindentation for soft materials.
After switching on the instrument and mounting the selected probe for the experiment, begin calibrating the probe. Click Initialize on the main software window. In the Calibration Menu that appears, enter the probe details in the input boxes.
Next, fill a thick, glass Petri dish with a flat bottom with the same medium as the sample dish, and match the temperature of the medium with that of the sample. Then, place the calibration dish under the probe. For calibration in liquid, pre-wet the probe with a drop of 70%ethanol or isopropanol with the end of the pipette in light contact with the glass ferrule, such that the drop slides over the cantilever and spherical tip.
Then, manually slide the nanoindenter's arm downwards until the probe is fully submerged but still far away from the bottom of the Petri dish. Wait for five minutes to allow equilibrium conditions to be reached in the liquid. Next, in the software's Initialize Menu, click on Scan Wavelength.
The interferometer's screen will show a progress bar. Check whether the optical scan was successful by navigating to the Wavelength Scan panel on the interferometer box. Then, in the Initialize Menu, click on Find Surface to progressively lower the probe.
The probe stops moving when it contacts the glass Petri dish. Once the probe is in contact with the surface, move the probe down by one micrometer using the y downwards arrow button on the main software window. Observe the green signal in the live window for changes in the baseline with each one-micrometer step.
Then, click Calibrate from the Initialize Menu. When the calibration is complete, check the old and new calibration factors on the popup window displayed. If the new calibration factor is in the correct range, as explained in the manuscript, click on Use New Factor.
Next, move up the piezo by 500 micrometers. Then, check whether the demodulation circle has been correctly calibrated by navigating to the Demodulation tab on the interferometer desktop. Gently tap on the optical table or the nanoindenter to induce enough noise.
A white circle of discrete data points should approximately cover the red circle. Load the Petri dish containing the sample on the microscope stage, and manually move the nanoindenter's probe to a desired position above the sample. Slide the probe in solution, taking care to leave one to two millimeters between the probe and the sample's surface.
Wait five minutes for the probe to equilibrate in solution. Focus on the probe with the optical microscope. To measure the Young's modulus of soft materials, click on Configure Experiment.
Add a Find Surface step and a single indentation in displacement control to determine experimental parameters to subsequently use for the automatic matrix scan. If the single indentation is successful, configure a matrix scan containing 50 to 100 points spaced at 10 to 100 micrometers. After ensuring that the Auto Find Surface box is ticked, click on Use Stage Position to start the matrix scan from the current stage position.
Set up the matrix scan profile in displacement control. Leave the number of segments to five, and use the default displacement profile. If necessary, change the displacement profile and time for each sloped segment.
Do not exceed strain rates greater than 10 micrometers per second. Save the configured experiment in the desired experiment path. Click on Run Experiment, and wait for it to be completed.
When all data is acquired, clean the probe and switch off the instrument as described in the text. For screening of force-displacement curves and production of cleaned data set in JSON format, launch prepare. py from the command line on the lab computer.
Select the Optics11 data format from the dropdown list. If the data is not loaded correctly, relaunch the graphical user interface and select Optics11 Old. Then, click on Load Folder, and select a folder containing the data to be analyzed.
Clean the data set using the tabs present on the right of the graphical user interface. Then, click on Save JSON, and enter an appropriate name for the cleaned data set. Send the JSON file to the computer where the NanoAnalysis software was installed, if different from the current computer.
Launch the nano. py file from the command line. On the top left of the graphical user interface, click on Load Experiment and select the JSON file.
This will populate the file list and the raw curves graph showing the data set in terms of force-displacement curves. In the Stats box, check the values of the three parameters, N activated, N failed, and N excluded. To visualize a specific curve in more detail, click on the curve.
This will highlight it in green and show it on the current curve graph. Once a single curve has been selected, the R and k parameters will be populated in the Stats box. Once the data set has been further cleaned, filter any noise in the curves using the filters implemented in the Filtering box.
Then, inspect the filtered curves in the current curve graph. The filtered curve is in black, whereas the non-filtered version is green. To find the contact point, from the Contact Point box, choose one of a series of numerical procedures that have been implemented in the software.
Adjust the algorithm's parameters to suit the data set so that the contact point is located correctly, as explained in the manuscript. To view where the contact point has been found on a single curve, select the curve by clicking on it. Then, click Inspect.
Check the popup window that appears to identify where the contact point has been located. Next, click on Hertz Analysis. This will generate three graphs.
Check force indentation data for each curve in the data set, together with the average Hertz fit shown in red. Then, check the average force indentation curve with an error band showing one standard deviation, together with the average Hertz fit shown in red. Next, check the scatter plot of the Young's modulus originating from fitting the Hertz model to each individual curve.
Inspect the Results box for the computed mean Young's modulus and its standard deviation, and ensure they are reasonable for the given experiment. Then, in the Save box, click on Hertz. In the popup window, enter the file name and directory, and click on Save.
A tsv file will be created. Open the tsv file in any additional software for statistical analysis and further plotting. For cell nanoindentation data, click on Elasticity Spectra Analysis.
Inspect the two plots produced, namely Young's modulus as a function of the indentation depth for each curve and the average Young's modulus as a function of indentation fitted by a bilayer model. Once the analysis is finished, click on ES in the Save box. This will export a tsv file in the specified directory, which can be opened and plotted in any other software of choice.
A successful experiment results in the approach segment of a force-displacement curve, having a clear, flat baseline, a transition region, and a sloped region. Curves showing alterations from this shape are easily removed from the data set using NanoPrepare. Average force-indentation curves together with the average Hertz model for a soft polyacrylamide hydrogel and a stiff hydrogel are shown here.
By plotting individual values of the Young's modulus, the expected average Young's modulus was retrieved for both hydrogels. For cell nanoindentation experiments, the average force-indentation curve and the corresponding average Hertz model demonstrate that the Hertz model does not fully capture the evolution of force with increasing indentation depth for cell nanoindentation experiments. The average elasticity spectra fitted up to an indentation of 200 nanometers are demonstrated here.
Average elasticity spectra start increasing at an indentation depth of 200 nanometers, indicating the contribution of a substrate to the probed apparent Young's modulus. Because of this, 200 nanometer was chosen as the fitting range for both the Hertz and the bilayer model. Fitting the bilayer model allows the extract more information about the cell's mechanical state, including the cell actin cortex thickness, cell actin cortex modulus, and cell bulk modulus, as explained in the main text.
A direct comparison between the Hertz model and the elasticity spectra approach in terms of Young's modulus distribution reveals overlapping distributions with comparable means, demonstrating the feasibility of the elasticity spectra approach. Accurately locating the contact point and keeping the chosen algorithm parameters consistent between data sets one wants to compare is paramount to obtaining reliable comparisons between samples. The method is of general applicability to quantify the local elastic properties of biological samples, including spheroids, organoids, tissues, and in general all soft matter.
