Frequency-dependent cellular microrheology with pyramidal atomic force microscopy probes

Atomic force microscopy (AFM) is widely used to measure the elastic properties of living cells, typically by extracting the Youngs (elastic) modulus from force-indentation curves acquired at a fixed loading rate. However, it is increasingly clear that cells relax applied stresses over time and exhibit significant frequency-dependent loss properties, a phenomenon which figures centrally in many biological problems. AFM has been employed to capture these dynamic mechanical properties via microscale frequency sweeps, where the AFM probe indents the sample in an oscillatory fashion over a range of frequencies and the oscillatory sample response is recorded. The relationship between dynamic stress and strain is then used to extract frequency-dependent microrheological properties. Despite the power of AFM oscillatory microrheology, the method remains surprisingly underutilized. This may be due to the traditional call for sphere-tipped AFM probes, which produce comparatively low indentation pressures yet are often expensive to purchase and difficult to fabricate. There remains a need for a framework in which standard blunt pyramidal AFM probes can be used "off the shelf" for oscillatory microrheology. In this study, we present such a framing. We derive expressions to extract rheological moduli from the data and explore practical experimental issues such as calibration and parameter optimization, validating the method using agarose hydrogel standards. Finally, we perform mechanical measurements on cultured cells treated with cytoskeletal inhibitors nocodazole and cytochalasin D, which yield rheological changes consistent with expected contributions of the corresponding cytoskeletal networks. Statement of SignificanceAtomic force microscopy (AFM) is often used in a force spectroscopy mode to measure an apparent elastic modulus at a single indentation rate, but in reality the mechanical behavior of cells is strongly rate-dependent, reflecting cellular viscoelasticity. While cellular viscoelastic properties can be measured by AFM oscillatory microrheology, the method traditionally calls for sphere-tipped probes, which are expensive to purchase and difficult to reproducibly fabricate. Fewer studies have used pyramidal AFM probes. Here, we examine this method in detail. We walk through data analysis expressions, compare force spectroscopy and frequency sweep measurements of a soft hydrogel, and discuss experimental setup. We then demonstrate measurement and interpretation of viscoelastic parameters in a model cell line treated with cytoskeletal inhibitors.