Microfabricated cantilevers for measurement of subcellular and molecular forces

Fauver, Mark; Dunaway, Dwayne; Lilienfeld, David; Craighead, Harold G.; Pollack, Gerald H.

doi:10.1109/10.686797

Cited by 82 publications

(46 citation statements)

References 14 publications

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“…The sarcomeres were isolated using glass microneedles that were also used for force measurements. The microneedles were produced with a vertical pipette puller (KOPF 720, David Kopf Inst, Tujunga) and calibrated by a cross-bending method (1), using pairs of microfabricated cantilevers of known stiffness as reference beams (17,37) (Fig. 1, A-C).…”

Section: Methodsmentioning

confidence: 99%

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

Pavlov

Novinger

Rassier

2009

American Journal of Physiology-Cell Physiology

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Pavlov I, Novinger R, Rassier DE. The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle. Am J Physiol Cell Physiol 297: C1211-C1219, 2009. First published August 26, 2009; doi:10.1152/ajpcell.00233.2009.-The goal of this study was to develop a system to experiment with sarcomeres mechanically isolated from skeletal muscles. Single myofibrils from rabbit psoas were transferred into a temperature-controlled (22°C or 15°C) experimental chamber, and sarcomeres were isolated using precalibrated glass microneedles that were pierced externally, adjacent to the Z-lines. The force produced during activation was measured by tracking the displacement of the microneedles, and the sarcomere and half-sarcomere changes were measured by continuously tracking the Z-lines and A-bands position during the experiments. Sarcomeres produced a stress (force/cross-sectional area) of 112.75 Ϯ 4.96 nN/m 2 (15°C) and 128.47 Ϯ 5.58 nN/m 2 (22°C) at lengths between 2.0 m and 2.4 m. The descending limb was fitted with linear regression for length between 2.4 m and 3.5 m, which provided an abscissa extrapolating to 3.87 m. The force-length relation was remarkably similar to a theoretical curve based on the degree of filament overlap. During sarcomere activation, we tracked the distance between the center of the A-band and the Z-lines. At lengths below 1.6 m, movements of A-band were not detected. A-band movements increased with length to achieve a maximum displacement of 59.40 Ϯ 10.1 nm from the center at 2.0 m-2.4 m. A-band displacement decreased linearly in sarcomere lengths between 2.6 m and 3.6 m. A technique for monitoring force and length in single sarcomeres isolated from myofibrils represents a reliable technique to evaluate contractile mechanisms at the most basic, intact level of muscle organization, opening the possibility to clarify longstanding issues in the field of muscle contraction.cross-bridges; force-length relation; myosin-actin interaction THE MECHANISMS OF STRIATED muscle contraction are commonly associated with the sliding of actin filaments induced by myosin cross-bridge rotation (27), which causes sarcomere shortening and force production. Consequently, the investigation of sarcomere mechanics has been an important focus of research in the muscle field for several years. The evaluation of sarcomere mechanics has been used for the proposal and refinement of cross-bridge models (18,27,39), to understand the relation between force and filament overlap (11-13, 19, 21, 21), to evaluate the effects of muscle stretch and shortening in force transients, sarcomere nonuniformity, and ATP hydrolysis (8,9,14,20,37,38), and to analyze the kinetics of force development and relaxation (10, 43).Sarcomere mechanics are commonly investigated in isolated muscle fibers, a preparation in which the behavior of sarcomeres is evaluated indirectly, based on the averaged striation spacing produced by the regular arrangement of the thick and thin filaments. However, each muscle fiber has millions of sa...

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Section: Methodsmentioning

confidence: 99%

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

Pavlov

Novinger

Rassier

2009

American Journal of Physiology-Cell Physiology

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“…Briefly, one end of a myofibril was attached either to the tip of a glass needle or, in some instances, to a nanofabricated tension transducer (6). The other end was mounted on the moving glass tip of the piezoelectric motor, which could impose linear length changes on the specimen.…”

Section: Experimental Apparatusmentioning

confidence: 99%

Fundamental step size in single cardiac and skeletal sarcomeres

Yakovenko

Blyakhman

Pollack

2002

American Journal of Physiology-Cell Physiology

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(10,21). We have carried out parallel measurements on single myofibrils from rabbit cardiac muscle and bumblebee flight muscle. Activated specimens were released or stretched with a motorimposed ramp, and the time course of length of individual sarcomeres was measured by projecting the image of the striations onto a linear photodiode array and tracking the spacing between A-band centroids. We confirmed the 5.4-nm step. With subnanometer precision, however, we find that this value is two times that of a more fundamental step size of 2.7 nm.Step sizes were always integer multiples of 2.7 nm, whether the length change was positive or negative. This value is equal to the linear repeat of actin monomers along the thin filament, a result that ties dynamic events to molecular structure and places narrow constraints on any proposed molecular mechanism. single myofibril; actin; optical microscopy; quantal shortening IN APPROACHING THE MECHANISM of motility and contraction, attention has turned toward the elementary molecular translation step. Although measurements in the past have yielded steps of broadly variable size, possibly because of challenging analytical obstacles (8,14,17), recent experiments with improved signals have shown steps on the order of 5-6 nm (20), and experiments on single myosin molecules translating along actin filaments have shown step size consistently of ϳ5.4 nm (10, 21). The value 5.4 nm is equal to the monomer repeat along a single actin strand (10,13,15,20,21).We have carried out parallel measurements on single sarcomeres. The single sarcomere model is useful because it preserves the natural filament lattice, yet cooperativity assures that molecular translation steps are not obscured (19). Previous myofibril experiments have shown that activated sarcomere shortening occurred in steps that were integer multiples of 2.7 nm (2). Because of noise, however, detectability was limited to steps larger than 4-5 nm, so we could not establish whether the implied 2.7-nm quantum actually existed.In the present experiments, we employed a differentially based algorithm that could suppress noise contributions sufficiently to bring the detection limit to subnanometer levels (18). With this high-resolution algorithm, we measured steps in activated sarcomeres both during shortening and during imposed stretch. Although the 5.4-nm step seen in single molecule studies is confirmed in this study, the myofibril reveals a more fundamental step size of half that value, or 2.7 nm. METHODS Myofibril PreparationIsolated myofibrils were prepared from rabbit left ventricular trabecular muscles as described previously (12). Briefly, thin strips of muscle tissue were dissected for storage in rigor/glycerol solution (50/50 by volume) for a minimum of 5 days at Ϫ20°C. To obtain single myofibrils, glycerinated strips were minced, and the pieces were further skinned in a 4°C rigor solution containing 0.5% Triton X-100 for 30 min. After being washed with fresh rigor solution, the tissue pieces were homogenized in a blender (Sorv...

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“…To do so, the forces acting on the microrobot must be measured. Force measurements could be achieved using capacitive force sensors [9], atomic force microscope (AFM) [10], piezoresistive cantilevers [11], or magnetic bead [12]. But such approaches are intrusive, and it is troublesome to use them with the microrobot in endovascular-like environments.…”

Section: Introductionmentioning

confidence: 99%

Vision-based force sensing of a magnetic microrobot in a viscous flow

Belharet

Folio

Ferreira

2014

2014 IEEE International Conference on Robotics and Automation (ICRA)

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Abstract-This paper has presented a new vision-based force-sensing framework that allows to characterize the forces applied on a magnetic microrobot in an endovascular-like environment. Especially, unlike common approaches used with optical microscopy where orthographic projection model are used, we consider in this paper the weak-perspective model. The proposed vision-based force characterization allows to retrieve the three dimensional (3D) translational velocities and accelerations of a microrobot viewed from a digital microscope. Hence, thanks to the dynamic model the external forces are estimated on-line. The framework was applied and validated for a magnetic microrobot navigating in a viscous flow. Experimental results in two different environments illustrate the efficiency of the proposed method.

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Microfabricated cantilevers for measurement of subcellular and molecular forces

Cited by 82 publications

References 14 publications

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

The mechanical behavior of individual sarcomeres of myofibrils isolated from rabbit psoas muscle

Fundamental step size in single cardiac and skeletal sarcomeres

Vision-based force sensing of a magnetic microrobot in a viscous flow

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