Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Abstract

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (μm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferruletop optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Vitronectin-based hydrogels recapitulate neuroblastoma growth conditions

“…The selected cantilever had a stiffness of 0.46 Nm −1 and held a spherical tip of 52 µm radius. The collected curves were analyzed using a custom Python code ( Ciccone et al, 2022 ). Curves were first aligned using a baseline detection method based on the histogram of the force signal ( Duanis-Assaf, Razvag and Reches, 2019 ) and the corresponding indentation was calculated for each curve.…”

Patient's dermal fibroblasts as disease markers for visceral myopathy

“…Single cell stiffness measurements were performed using a Chiaro system (Optics11Life, NL), a nanoindentation device based on a ferruletop with interferometric read-out that enables high resolution force measurements in liquid environment [27]. The experimental protocol to operate the Chiaro nanoindenter is very similar to what is required for the more widely adopted atomic force microscope (AFM) and the data can be analyzed through the same approach [27,28]. In brief, the force is measured while moving the tip towards the sample and the corresponding F(Z) curve is recorded.…”

“…The mechanical properties of the cells were calculated by fitting the indentation curve with the Hertz model [29,30] up to a maximum indentation of 300 nm, selected to be smaller than 10 % of the thickness of the cell [31]. To calculate the contact-point we used a threshold method [28], keeping the same parameters for all datasets. The analysis was performed using a dedicated python software which is free and available under open source license [32] (https://github.com/CellMechLab/nanoindentat ion.git).…”

The Impact of Experimental Conditions on Cell Mechanics as Measured with Nanoindentation

“…The de facto standard for measuring single-cell mechanics is nanoindentation [ 8 ], either using an Atomic Force Microscope (AFM) [ 9 ], eventually with a colloidal probe [ 10 ], or more dedicated devices with different deflection detection methods [ 11 ]. The experimental procedure to perform nanoindentation and analyze the data is well established [ 12 ]. Nevertheless, obtaining robust and reproducible mechanical characterization of single cells is still a challenging task.…”