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.
Highlights
The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM)
The fluidic force microscope (FluidFM) system was created based on the principles of the atomic force microscope (AFM)[1]: a nanofluidic channel is attached to a refillable fluid reservoir, which is introduced into an AFM cantilever regulated by a pressure control system[2].The special microfabrication technology[3] of these hollow cantilevers allows the user to dispense or collect fluids in the femtoliter scale enabling wide functionallity[4]
(1) Does the special structure of the FluidFM cantilevers has any effect on the calibration of the spring constant or lever sensitivity? Since both of these parameters are measured with the laser beam reflection based optical detector, any differences compared to classical AFM cantilevers are expected to be discernable in this context, which leads to the few questions
Summary
The fluidic force microscope (FluidFM) can be considered as the nanofluidic extension of the atomic force microscope (AFM). During SCFS a characteristic force-distance (F-D) curve can be obtained from which properties such as the maximal adhesion force, adhesion energy and step-like events displaying the rupture of individual receptor-ligand interactions anchoring the cells to the substrate can be calculated[20] This type of spectroscopic data carries information regarding the cellular state, and the cells’ attachment to engineered or natural surfaces may reflect on their behavior represented by the F-D curves. The FluidFM micropipette cantilever has a similar shape to flat AFM cantilevers, by using the appropriate model for fitting (e.g. Herz-Sneddon model25) it can be utilized for the same purpose It is well-known in classical AFM methodology, that the precise calibration of the spring constant[25,26] and optical lever sensitivity[24] of the used cantilever is essential to gain reliable data with the above mentioned methods. Using the polystyrene sample holder plate for sensitivity measurements would be more convenient, since the elastic modulus of polystyrene is nearly two orders of magnitude smaller than that of silicon, it is rightful to ask that, (5) would using a polystyrene plate instead of silicon introduce any significant error during the calibration of lever sensitivity?
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