Elsevier

Journal of Biomechanics

Volume 48, Issue 6, 13 April 2015, Pages 1099-1104
Journal of Biomechanics

Toward the realization of reproducible Atomic Force Microscopy measurements of elastic modulus in biological samples

https://doi.org/10.1016/j.jbiomech.2015.01.023Get rights and content

Abstract

The validation of the AFM method for elastic modulus E measurement in soft materials (E <5 MPa) is still missing. The interest of measurements in materials with E <5 MPa is mainly biological, including soft tissues and single cells. For the diagnosis of malignant human tumors, a change in cell elasticity, within tissues, has recently been recognized as a marker of metastatic potential. To measure a cell elasticity difference, reproducible E measurements in biological samples are needed. In this work a robust method for a metrological validation of E measurements in the range 500–5000 kPa was developed, based on the realization of thick E standard samples and on the study of the interactions between the measurement process and the sample at micro- and nano-scale. E measurement reproducibility limit of 4% has been reached. This allows designing a very sensitive and reproducible measurement of E in biological samples representing thus a powerful diagnostic tool for cancer detection.

Introduction

Atomic Force Microscopy (AFM) allows high-resolution imaging of biological samples and the characterization of mechanical properties of very soft and non-homogeneous materials, such biological samples, by detecting repulsive and attractive cell surface forces (Cross et al., 2007, Kuznetsova et al., 2007). Young׳s modulus or elastic modulus (E) is a measure of materials stiffness; it can be measured by AFM (Kuznetsova et al., 2007, Darling et al., 2007, Costa, 2004) and gives information on biological sample (e.g. single cell within a tissue) elasticity.

The validation of the AFM method for E measurement in materials with E<5 MPa is still missing (Carrillo et al., 2005). In the low range, the E measurement by AFM is influenced by the interaction between the measurement system and the material of which E is measured. Therefore, a metrological characterization of the system interaction needs to be determined. The interest of E measurements in materials with E<5 MPa is mainly biological: soft tissues and single cells or cell cultures exhibit E in this range (Wenger et al., 2007).

Recently, a change in cell E has been recognized as a marker of disease such as cancer (Cross et al., 2007, Cross et al., 2008, Guo et al., 2012). Changes in the extracellular matrix and cytoskeleton structure have been found translating into cell elasticity changes (Bhadriraju and Hansen, 2002). In 2007, Cross et al. found a difference in E between living human metastatic cancer cells and the corresponding benign cells; they measured by AFM that malignant cells are 70% softer than benign cells. Current and traditional analyses for cancer cell detection (such as cytomorphological and immunohistochemical analyses) (Lekka et al., 2012a, Lekka et al., 2012b) are qualitative morphological analysis; they relies on cytoskeleton remodeling leading to cell shape changes. However traditional methods for malignant cells diagnosis have a limitation; frequent morphological overlap between tumor and normal cell types occurs (Cross et al., 2007). Cross et al. also demonstrated that AFM measurements of E well-correlate with traditional methods of cancer cell detection. Therefore, AFM mechanical analysis offers the powerful tool to quantitatively distinguish malignant cells from normal cells for cancer detection. To measure a cell elasticity difference, reproducible elasticity measurements of the biological sample are needed and the target reproducibility must be lower than the expected cell elasticity difference (70%). As a consequence the measurement method, AFM Force Spectroscopy, must be validated for reproducibility.

Investigation for cancer detection can involve single cells (Lekka et al., 2012a, Lekka et al., 2012b, Li et al., 2008) and tissues (Lekka et al., 2012b) coming from biopsies. Consequently, investigations should cover measures at macro-, micro- and nanoscale, respectively for analyzing the extended E in a tissue, the specific E of a single cell and also E of defined cells substructures at nanoscale. It has been shown (Lekka et al., 2012a, Lekka et al., 2012b) that E measured at single cell level and tissue level (nano-, micro- and macro- levels) can be different, and the combination of the two AFM measurements offers a precious set of information about cancer detection. To perform reproducible E measurement on different biological samples (tissues, single cell, cells substructures) the AFM method must be validated in different scale ranges. In addition, high indentation speeds must be tested in order to perform measurements in time limits compatible with cellular processes of living cells such as cell mobility (lifespan: seconds) and cell division, apoptosis (lifespan: minutes).

With this work a robust method for a metrological validation of E measurements in the range 500–5000 kPa was developed, based on the realization of thick samples showing a homogeneous E value on macro-, micro- and nanoscale, and on the study of the interactions between the measurement process and the sample. Sylgard 184 was chosen as modeling material for soft tissues, as also described in our previous work (Demichelis, 2013, Demichelis et al., 2014). Sylgard samples in biological elastic range of 50–5000 kPa were prepared. Indentations with the AFM sensor were performed to characterize surface homogeneity and viscoelastic behavior of samples. Its use as multiscale standard was also investigated. Operative measurement settings were obtained for the realization of reproducible elasticity measurements on biological materials.

Results obtained in this work will allow designing a very sensitive and reproducible measurement of E in biological samples aimed in measuring elasticity differences below 5%.

Section snippets

Sylgard as E standard in the range 500–5000 kPa

The validation of the AFM Force Spectroscopy method on soft materials requires E standards. The standard must have an E defined in all its volume, must present homogeneity properties and stability over time. Procedures for preparation of standards must be defined; they can invalidate the employ of the standard since influence the sample homogeneity in all directions, both xy plane and z direction.

Polydimethylsiloxane PDMS is a viscoelastic polymer of cross-linked chains that can be prepared

Preparation of Sylgard samples

Fresh Sylgard 184 (Dow Corning) rectangular samples, 0.5 cm height, were realized in a grid plastic stamp, with nominal base/curing ratio of 15, 25 and 55 by weight. Stirring time of 2 min and curing time of 24 h were set. The stamp was put on the AFM stage and each compartment was filled with deionized water, for AFM measurements in liquid. The employed storing method consisted of storing the samples at room temperature, without water on the surface, covering them with a plastic cup, washing the

E function of indentation speed

The following results can be derived by Fig. 1:

  • Sylgard 1:15,1:25, and 1:55 present E of round 2000, 500, and 50 kPa respectively, measured with different indenters at different scales.

  • For Sylgard 1:15 and 1:25 a 10% variation of E is observed in the range 0.1–100 µm/s with a plateau region around 5 µm/s. For Sylgard 1:55 the plateau is not easily determined. After 10, 100, and 200 µm/s for respectively Sylgard 1:15, 1:25, and 1:55 a significant decrease of E is observed.

  • The E function of

Discussion

The E realized from Sylgard 15, 25 and 55 are consistent with the target range below 5 MPa, are stable over time and maintained at micro- and nanoscale with different indenters. It follows that Sylgard 184 can be considered a suitable material to realize elasticity standards.

The variation of elasticity in a range 0.1–100 µm/s is contained in the measurement reproducibility, where an indentation speed of 5 µm/s can be considered optimal to measure the elasticity behavior of PDMS sample in the range

Conclusion

In this work, a robust method for the validation of E measurements in the biological range between 500–5000 kPa was proposed. It is based on the realization of thick E standards, identification of conditions for reproducible measures of standards and maintaining of the same measurement setting on biological samples. A method for the calculation with AFM of the E variability in a given sample layer is given. Standards at 2000, 500, and 50 kPa were realized in a range not currently available. For

Conflict of interest

The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers׳ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patentlicensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed

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