Eliminating mechanical perturbations in scanning probe microscopy

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.

Section: Atomic Force Microscopy For Biophysical Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differential force microscope for long time-scale biophysical measurements

Choy

Parekh

Chaudhuri

et al. 2007

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Weafer

McGarry

et al. 2012

Atomic force microscopy (AFM) is widely used in the study of both morphology and mechanical properties of living cells under physiologically relevant conditions. However, quantitative experiments on timescales of minutes to hours are generally limited by thermal drift in the instrument, particularly in the vertical (z) direction. In addition, we demonstrate the necessity to remove all air-liquid interfaces within the system for measurements in liquid environments, which may otherwise result in perturbations in the measured deflection. These effects severely limit the use of AFM as a practical tool for the study of long-term cell behavior, where precise knowledge of the tip-sample distance is a crucial requirement. Here we present a readily implementable, cost effective method of minimizing z-drift and liquid instabilities by utilizing active temperature control combined with a customized fluid cell system. Long-term whole cell mechanical measurements were performed using this stabilized AFM by attaching a large sphere to a cantilever in order to approximate a parallel plate system. An extensive examination of the effects of sphere attachment on AFM data is presented. Profiling of cantilever bending during substrate indentation revealed that the optical lever assumption of free ended cantilevering is inappropriate when sphere constraining occurs, which applies an additional torque to the cantilevers “free” end. Here we present the steps required to accurately determine force-indentation measurements for such a scenario. Combining these readily implementable modifications, we demonstrate the ability to investigate long-term whole cell mechanics by performing strain controlled cyclic deformation of single osteoblasts.

“…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. However, this approach does not prevent cantilever bending against a stationary surface while the cantilever is connected to the surface through a biomolecule or a cell.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Torun

Finkler

Degertekin

2009

The authors describe a method for athermalization in atomic force microscope ͑AFM͒ based force spectroscopy applications using microstructures that thermomechanically match the AFM probes. The method uses a setup where the AFM probe is coupled with the matched structure and the displacements of both structures are read out simultaneously. The matched structure displaces with the AFM probe as temperature changes, thus the force applied to the sample can be kept constant without the need for a separate feedback loop for thermal drift compensation, and the differential signal can be used to cancel the shift in zero-force level of the AFM.