Young's modulus mapping by AFM

Standard bioAFM microscope JPK NanoWizard 3 (JPK, Berlin, Germany) was used to perform force mapping procedure. The scanning-by-probe head (maximal visualization range 100-100-15 μm in X-Y-Z axis) of AFM microscope was placed on inverted optical microscope Olympus IX-81, × 10 objective was used to find proper area covered with cells and to place cantilever in the proper position for force mapping procedure. Plastic Petri dish (TPP, Trasadingen, Switzerland) with either the distilled water for instrument calibration or with cell culture was placed inside the Petri dish heater (JPK) pre-heated to 37 °C for 30 min. Non-coated silicon nitride AFM cantilever Hydra 2R-100N (AppNano, California, USA) equipped with pyramidal silicon tip was used for all the experiments. The probe was calibrated before each experiment as described below. Then the laser reflection sum was maximized, followed by centering of the laser detector. The AFM probe was introduced in contact with the surface during a standard process of landing. The sensitivity of the AFM setup was determined as a slope of the force-distance curve (FDC) measured by lifting the cantilever with Z-height of 450 nm, time per curve was 3 s. The sensitivity was found in the range 15.07-15.37 nm V⁻¹, cantilever stiffness was calibrated by measurement of its thermal noise and resulted between 17.34 and 19.19 pN nm⁻¹ for different days of experiments.

The bioAFM setting was identical for all the force mapping procedure. SetPoint value was 1.0 nN (relative to baseline value), time per curve 0.5 s, Z-length 15.0 μm, speed of curve recording 30 μm s⁻¹, the FDCs were recorded with data sample rate of 2 kHz. The force mapping procedure was performed as step-by-step recording of FDCs in the network of 64 × 64 points on 100 × 100 μm covering area of single cell for AD-MSCs or colonies for CAL51. Force mapping process provides a network of FDC (dependency of tip-sample interaction force on tip height above the surface), so called force maps. The absolute value of Young's modulus can be determined by fitting the FDC by Sneddon equation⁵⁴:

Where F is the measured force, E is Young's modulus, ν is Poisson ration (0.5 for incompressible materials), δ is tip-sample separation (obtained by correction of the cantilever height to its bending) and α is half-angle to face of pyramidal tip (reflects the tip geometry). Data processing module of the JPK software was used to process the maps of FDCs in a batch mode. Resulted Young's modulus maps were exported to be post-processed as described in the following chapter.