Differential force microscope for long time-scale biophysical measurements

Abstract

Force microscopy techniques including optical trapping, magnetic tweezers, and atomic force microscopy (AFM) have facilitated quantification of forces and distances on the molecular scale. However, sensitivity and stability limitations have prevented the application of these techniques to biophysical systems that generate large forces over long times, such as actin filament networks. Growth of actin networks drives cellular shape change and generates nano-Newtons of force over time scales of minutes to hours, and consequently network growth properties have been difficult to study. Here, we present an AFM-based differential force microscope with integrated epifluorescence imaging in which two adjacent cantilevers on the same rigid support are used to provide increased measurement stability. We demonstrate 14 nm displacement control over measurement times of 3 hours and apply the instrument to quantify actin network growth in vitro under controlled loads. By measuring both network length and total network fluorescence simultaneously, we show that the average cross-sectional density of the growing network remains constant under static loads. The differential force microscope presented here provides a sensitive method for quantifying force and displacement with long time-scale stability that is useful for measurements of slow biophysical processes in whole cells or in reconstituted molecular systems in vitro.

Ultrastable atomic force microscopy: Improved force and positional stability

“…Interferometric stabilization is often used in high-precision physics experiments, but, in AFM applications, such stabilization requires affixing a diffraction grating to the back of the cantilever holder and the sample [63]. A handful of techniques have been proposed [30,62,64] to combat vertical drift during imaging. In one innovative design [64], an AFM containing two independently steerable optical-lever-arm detection systems measured a chip containing two different length AFM cantilevers.…”

“…A handful of techniques have been proposed [30,62,64] to combat vertical drift during imaging. In one innovative design [64], an AFM containing two independently steerable optical-lever-arm detection systems measured a chip containing two different length AFM cantilevers. The longer cantilever enabled vertical stabilization of the sample relative to the cantilever holder.…”

Stability enhancement of an atomic force microscope for long-term force measurement including cantilever modification for whole cell deformation

“…8,9,10 Approaches including intermittent substrate referencing between force measurements 11 or the use of reference sensors 12,13,14,15 can be applied to counteract, rather than prevent, thermal drift in the vertical direction (z-drift). However, intermittent substrate referencing is unsuitable when the cell dimensions exceed the lateral range of the AFM scanner or when continuous contact between the sphere and cell is a requirement.…”

Athermalization in atomic force microscope based force spectroscopy using matched microstructure coupling

“…14 Maintaining the set peak force without the need for an external driver or feedback is a unique capability with the introduced approach when compared to the previously demonstrated methods. 4,[10][11][12] Note that the cantilever still bends, and there is a shift in zero-force level set for the cantilever. This can be corrected by reading the displacement of the membrane.…”

“…When the cantilever should be engaged on the sample for the entire experiment, the referencing can be done by reading the deflection of a reference sensor. The reference sensor, which provides distance information from the cantilever substrate-to-sample can simply be another cantilever next to the measurement one, 10,11 an interferometer, 12 or an electrostatic sensor. 4 The reference sensor provides information for compensation of drift in distance from cantilever plane to sample substrate.…”