title: Quantifying Force and Viscoelasticity Inside Living Cells Using an Active-Passive Calibrated Optical Trap.
abstract: As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid.In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often for precise quantitative force measurements, due to the unknown value of the spring constant of the optical trap formed within the cell's viscoelastic cytoplasm. Here, we describe a method for utilizing optical tweezers for quantitative in vivo force measurements. The experimental protocol and the protocol for data analysis rely on two types of experiments, passive observation of the thermal motion of a trapped object inside a living cell, followed by observations of the response of the trapped object when subject to controlled oscillations of the optical trap. One advantage of this calibration method is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S. pombe cells.
authors.affiliation: Niels Bohr Institute, University of Copenhagen, Blegdamsvej 17, 2100, Copenhagen, Denmark.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Ritter
authors.affiliation: School of Physics & Astronomy, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9SS, Scotland, UK.
authors.forename: Josep
authors.initials: J
authors.lastname: Mas
authors.affiliation: Niels Bohr Institute, University of Copenhagen, Blegdamsvej 17, 2100, Copenhagen, Denmark.
authors.forename: Lene
authors.initials: L
authors.lastname: Oddershede
authors.affiliation: Department of Physics, Technical University of Denmark, Building 309, 2800, Kongens Lyngby, Denmark. kirstine.berg-sorensen@fysik.dtu.dk.
authors.forename: Kirstine
authors.initials: K
authors.lastname: Berg-Sørensen
mesh_terms: D002138:Calibration; D004548:Elasticity; D008853:Microscopy; D052898:Optical Tweezers; D055095:Optics and Photonics / Q000379:methods*; D012568:Schizosaccharomyces; D014783:Viscosity
keywords: Cytoplasm; Force measurements; In vivo; Optical tweezers; Spring constant; Viscoelasticity