Dataset Information

Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

ABSTRACT: The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM). This novel instrument facilitates the experimental procedure and data acquisition of force spectroscopy (FS) and is also used for the determination of single-cell adhesion forces (SCFS) and elasticity. FluidFM uses special probes with an integrated nanochannel inside the cantilevers supported by parallel rows of pillars. However, little is known about how the properties of these hollow cantilevers affect the most important parameters which directly scale the obtained spectroscopic data: the inverse optical lever sensitivity (InvOLS) and the spring constant (k). The precise determination of these parameters during calibration is essential in order to gain reliable, comparable and consistent results with SCFS. Demonstrated by our literature survey, the standard error of previously published SCFS results obtained with FluidFM ranges from 11.8% to 50%. The question arises whether this can be accounted for biological diversity or may be the consequence of improper calibration. Thus the aim of our work was to investigate the calibration accuracy of these parameters and their dependence on: (1) the aperture size (2, 4 and 8?µm) of the hollow micropipette type cantilever; (2) the position of the laser spot on the back of the cantilever; (3) the substrate used for calibration (silicon or polystyrene). It was found that both the obtained InvOLS and spring constant values depend significantly on the position of the laser spot. Apart from the theoretically expectable monotonous increase in InvOLS (from the tip to the base of the cantilever, as functions of the laser spot's position), we discerned a well-defined and reproducible fluctuation, which can be as high as ±30%, regardless of the used aperture size or substrate. The calibration of spring constant also showed an error in the range of -13/+20%, measured at the first 40?µm of the cantilever. Based on our results a calibration strategy is proposed and the optimal laser position which yields the most reliable spring constant values was determined and found to be on the first pair of pillars. Our proposed method helps in reducing the error introduced via improper calibration and thus increases the reliability of subsequent cell adhesion force or elasticity measurements with FluidFM.

SUBMITTER: Nagy AG

PROVIDER: S-EPMC6635487 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

Nagy Ágoston G ÁG Kámán Judit J Horváth Róbert R Bonyár Attila A

Scientific reports 20190716 1

The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM). This novel instrument facilitates the experimental procedure and data acquisition of force spectroscopy (FS) and is also used for the determination of single-cell adhesion forces (SCFS) and elasticity. FluidFM uses special probes with an integrated nanochannel inside the cantilevers supported by parallel rows of pillars. However, little is known about how the properties of ...[more]

PMID: 31311966

Similar Datasets

Project description:Flagella and cilia are cellular appendages that inherit essential functions of microbial life including sensing and navigating the environment. In order to propel a swimming microorganism they displace the surrounding fluid by means of periodic motions, while precisely timed modulations of their beating patterns enable the cell to steer towards or away from specific locations. Characterizing the dynamic forces, however, is challenging and typically relies on indirect experimental approaches. Here, we present direct in vivo measurements of the dynamic forces of motile Chlamydomonas reinhardtii cells in controlled environments. The experiments are based on partially aspirating a living microorganism at the tip of a micropipette force sensor and optically recording the micropipette's position fluctuations with high temporal and sub-pixel spatial resolution. Spectral signal analysis allows for isolating the cell-generated dynamic forces caused by the periodic motion of the flagella from background noise. We provide an analytic, elasto-hydrodynamic model for the micropipette force sensor and describe how to obtain the micropipette's full frequency response function from a dynamic force calibration. Using this approach, we measure the amplitude of the oscillatory forces during the swimming activity of individual Chlamydomonas reinhardtii cells of 26 ± 5 pN, resulting from the coordinated flagellar beating with a frequency of 49 ± 5 Hz. This dynamic micropipette force sensor technique generalizes the applicability of micropipettes as force sensors from static to dynamic force measurements, yielding a force sensitivity in the piconewton range. In addition to measurements in bulk liquid environment, we study the dynamic forces of the biflagellated microswimmer in the vicinity of a solid/liquid interface. As we gradually decrease the distance of the swimming microbe to the interface, we measure a significantly enhanced force transduction at distances larger than the maximum extent of the beating flagella, highlighting the importance of hydrodynamic interactions for scenarios in which flagellated microorganisms encounter surfaces.

Project description:As the application of atomic force microscopy (AFM) in soft matter characterization has expanded, the use of different types of cantilevers for these studies have also increased. One of the most common types of cantilevers used in soft matter imaging is V-shaped cantilevers due to their low normal spring constant. These types of cantilevers are also suitable for nanomanipulation due to their high lateral spring constants. The combination of low normal spring constant and high lateral spring constants makes V-shaped cantilevers promising candidates for imaging soft matter. Although these cantilevers are widely used in the field, there are no studies on the static and dynamic behavior of V-shaped cantilevers in multifrequency AFM due to their complex geometry. In this work, the static and dynamic properties of V-shaped cantilevers are studied while investigating their performance in multifrequency AFM (specifically bimodal AFM). By modeling the cantilevers based on Timoshenko beam theory, the geometrical dimensions such as length, base width, leg width and thickness are studied. By finding the static properties (mass, spring constants) and dynamic properties (resonance frequencies and quality factors) for different geometrical dimensions, the optimum V-shaped cantilever that can provide the maximum phase contrast in bimodal AFM between gold (Au) and polystyrene (PS) is found. Based on this study, it is found that as the length of the cantilever increases the 2nd eigenmode phase contrast decreases. However, the base width exhibits the opposite relationship. It is also found that the leg width does not have a monotone relationship similar to length and base width. The phase contrast increases for the range of 14 to 32 µm but decreases afterwards. The thickness of a V-shaped cantilever does not play a major role in defining the dynamics of the cantilever compared to other parameters. This work shows that in order to maximize the phase contrast, the ratio of second to first eigenmode frequencies should be minimized and be close to a whole number. Additionally, since V-shaped cantilevers are mostly used for soft matter imaging, lower frequency ratios dictate lower spring constant ratios, which can be advantageous due to lower forces applied to the surface by the tip given a sufficiently high first eigenmode frequency. Finally, two commercially available V-shaped cantilevers are theoretically and experimentally benchmarked with an optimum rectangular cantilever. Two sets of bimodal AFM experiments are carried out on Au-PS and PS-LDPE (polystyrene and low-density polyethylene) samples to verify the simulation results.

Dataset Information

Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

Publications

Spring constant and sensitivity calibration of FluidFM micropipette cantilevers for force spectroscopy measurements.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets