TY - JOUR

T1 - Quantifying Force and Viscoelasticity Inside Living Cells Using an Active-Passive Calibrated Optical Trap

AU - Ritter, Christine M

AU - Mas, Josep

AU - Oddershede, Lene

AU - Berg-Sørensen, Kirstine

PY - 2017/3/20

Y1 - 2017/3/20

N2 - 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.

AB - 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.

KW - Optical tweezers

KW - Viscoelasticity

KW - Cytoplasm

KW - In vivo

KW - Force measurements

KW - Spring constant

U2 - 10.1007/978-1-4939-6421-5_20

DO - 10.1007/978-1-4939-6421-5_20

M3 - Journal article

C2 - 27844442

VL - 1486

SP - 513

EP - 536

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -