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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fazal FM, Block SM (2011) Optical tweezers study life under tension. Nat Photonics 5:318–321
Moffitt JR, Chemla YR, Smith SB et al (2008) Recent advances in optical tweezers. Annu Rev Biochem 77(1):205–228
Greenleaf WJ, Woodside MT, Block SM (2007) High-resolution, single-molecule measurements of biomolecular motion. Annu Rev Biophys Biomol Struct 36:171–190
Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5(6):491–505
Berg-Sørensen K, Flyvbjerg H (2004) Power spectrum analysis for optical tweezers. Rev Sci Instrum 75(3):594–612
Tolić-Nørrelykke SF, Schäffer E, Howard J et al (2006) Calibration of optical tweezers with positional detection in the back focal plane. Rev Sci Instrum 77(10):103101
Neuman KC, Block SM (2004) Optical trapping. Rev Sci Instrum 75(9):2787–2809
Gittes F, Schmidt CF (1998) Signals and noise in micromechanical measurements. Methods Cell Biol 55:129–156
Veigel C, Schmidt CF (2011) Moving into the cell: single-molecule studies of molecular motors in complex environments. Nat Rev Mol Cell Biol 12(3):163–176
Sims PA, Xie XS (2009) Probing dynein and kinesin stepping with mechanical manipulation in a living cell. ChemPhysChem 10(9-10):1511–1516
Leidel C, Longoria RA, Gutierrez FM et al (2012) Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport. Biophys J 103(3):492–500
Shubeita GT, Tran SL, Xu J et al (2008) Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets. Cell 135(6):1098–1107
Guet D, Mandal K, Pinot M et al (2014) Mechanical role of actin dynamics in the rheology of the Golgi complex and in Golgi-associated trafficking events. Curr Biol 24(15):1700–1711
Oddershede LB (2012) Force probing of individual molecules inside the living cell is now a reality. Nat Chem Biol 8(11):879–886
Norregaard K, Jauffred L, Berg-Sorensen K et al (2014) Optical manipulation of single molecules in the living cell. Phys Chem Chem Phys 16(25):12614–12624
López-Quesada C, Fontaine AS, Farré A et al (2014) Artificially-induced organelles are optimal targets for optical trapping experiments in living cells. Biomed Opt Express 5(7):1993–2008
Gross SP (2003) Application of optical traps in vivo. Methods Enzymol 361:162–174
Rasmussen MB, Oddershede LB, Siegumfeldt H (2008) Optical tweezers cause physiological damage to Escherichia coli and Listeria bacteria. Appl Environ Microbiol 74(8):2441–2446
Peterman EJG, Gittes F, Schmidt CF (2003) Laser-induced heating in optical traps. Biophys J 84(2):1308–1316
Neuman KC, Chadd EH, Liou GF et al (1999) Characterization of photodamage to Escherichia coli in optical traps. Biophys J 77(5):2856–2863
Lee YJ, Patel D, Park S (2011) Local rheology of human neutrophils investigated using atomic force microscopy. Int J Biol Sci 7(1):102–111
Wilhelm C (2008) Out-of-equilibrium microrheology inside living cells. Phys Rev Lett 101(2):028101
Dufrene YF, Evans E, Engel A et al (2011) Five challenges to bringing single-molecule force spectroscopy into living cells. Nat Methods 8(2):123–127
Jun Y, Tripathy SK, Narayanareddy BR et al (2014) Calibration of optical tweezers for in vivo force measurements: how do different approaches compare? Biophys J 107(6):1474–1484
Tolić-Nørrelykke IM, Munteanu E-L, Thon G et al (2004) Anomalous diffusion in living yeast cells. Phys Rev Lett 93(7):078102
Schnurr B, Gittes F, MacKintosh FC et al (1997) Determining microscopic viscoelasticity in flexible and semiflexible polymer networks from thermal fluctuations. Macromolecules 30(25):7781–7792
Gittes F, Schnurr B, Olmsted PD et al (1997) Microscopic viscoelasticity: shear moduli of soft materials determined from thermal fluctuations. Phys Rev Lett 79(17):3286–3289
Mas J, Farré A, López-Quesada C et al (2011) Measuring stall forces in vivo with optical tweezers through light momentum changes. Proc SPIE 8097:809726
Farré A, Montes-Usategui M (2010) A force detection technique for single-beam optical traps based on direct measurement of light momentum changes. Opt Express 18(11):11955–11968
Farré A, Marsà F, Montes-Usategui M (2012) Optimized back-focal-plane interferometry directly measures forces of optically trapped particles. Opt Express 20(11):12270–12291
Smith SB, Cui Y, Bustamante C (2003) Optical-trap force transducer that operates by direct measurement of light momentum. In: Methods in Enzymology. Academic, New York, NY, pp 134–162
Hendricks AG, Holzbaur EL, Goldman YE (2012) Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors. Proc Natl Acad Sci U S A 109(45):18447–18452
Vermeulen KC, van Mameren J, Stienen GJM et al (2006) Calibrating bead displacements in optical tweezers using acousto-optic deflectors. Rev Sci Instrum 77(1):013704
Blehm BH, Schroer TA, Trybus KM et al (2013) In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport. Proc Natl Acad Sci 110(9):3381–3386
Valentine MT, Guydosh NR, Gutiérrez-Medina B et al (2008) Precision steering of an optical trap by electro-optic deflection. Opt Lett 33(6):599–601
Mas J, Richardson AC, Reihani SN et al (2013) Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells. Phys Biol 10(4):046006
Fischer M, Richardson AC, Reihani SN et al (2010) Active-passive calibration of optical tweezers in viscoelastic media. Rev Sci Instrum 81(1):015103
Fischer M, Berg-Sorensen K (2007) Calibration of trapping force and response function of optical tweezers in viscoelastic media. J Opt A Pure Appl Opt 9(8):S239–S250
Atakhorrami M, Sulkowska JI, Addas KM et al (2006) Correlated fluctuations of microparticles in viscoelastic solutions: Quantitative measurement of material properties by microrheology in the presence of optical traps. Phys Rev E 73(6):061501
Mizuno D, Head DA, MacKintosh FC et al (2008) Active and passive microrheology in equilibrium and nonequilibrium systems. Macromolecules 41(19):7194–7202
Lee H, Ferrer JM, Nakamura F et al (2010) Passive and active microrheology for cross-linked F-actin networks in vitro. Acta Biomater 6(4):1207–1218
Lau AWC, Hoffman BD, Davies A et al (2003) Microrheology, stress fluctuations, and active behavior of living cells. Phys Rev Lett 91(19):198101
Robert D, Nguyen T-H, Gallet F et al (2010) In vivo determination of fluctuating forces during endosome trafficking using a combination of active and passive microrheology. PLoS One 5(4):e10046
Andersson M, Czerwinski F, Oddershede LB (2011) Optimizing active and passive calibration of optical tweezers. J Opt 13(4):044020
Oddershede L, Greco S, Nørrelykke SF et al (2001) Optical tweezers: probing biological surfaces. Probe Microsc 2:129
Gittes F, Schmidt CF (1998) Interference model for back-focal-plane displacement detection in optical tweezers. Opt Lett 23(1):7–9
Pralle A, Prummer M, Florin EL et al (1999) Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light. Microsc Res Tech 44(5):378–386
Leijnse N, Jeon JH, Loft S et al (2012) Diffusion inside living human cells. Eur Phys J Spec Top 204(1):75–84
Dreyer JK, Berg-Sorensen K, Oddershede L (2004) Improved axial position detection in optical tweezers measurements. Appl Opt 43(10):1991–1995
Richardson AC, Reihani SNS, Oddershede LB (2008) Non-harmonic potential of a single beam optical trap. Opt Express 16(20):15709–15717
Mizuno D, Tardin C, Schmidt CF et al (2007) Nonequilibrium mechanics of active cytoskeletal networks. Science 315(5810):370–373
Mizuno D, Bacabac R, Tardin C et al (2009) High-resolution probing of cellular force transmission. Phys Rev Lett 102(16):168102
Ott D, Reihani SN, Oddershede LB (2014) Crosstalk elimination in the detection of dual-beam optical tweezers by spatial filtering. Rev Sci Instrum 85(5):053108
Jeon J-H, Tejedor V, Burov S et al (2011) In vivo anomalous diffusion and weak ergodicity breaking of lipid granules. Phys Rev Lett 106(4):048103
Selhuber-Unkel C, Yde P, Berg-Sorensen K et al (2009) Variety in intracellular diffusion during the cell cycle. Phys Biol 6(2):025015
Yamada S, Wirtz D, Kuo SC (2000) Mechanics of living cells measured by laser tracking microrheology. Biophys J 78(4):1736–1747
Wei M-T, Zaorski A, Yalcin HC et al (2008) A comparative study of living cell micromechanical properties by oscillatory optical tweezers. Opt Express 16(12):8594–8603
Czerwinski F, Richardson AC, Oddershede LB (2009) Quantifying noise in optical tweezers by allan variance. Opt Express 17(15):13255–13269
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ritter, C.M., Mas, J., Oddershede, L., Berg-Sørensen, K. (2017). Quantifying Force and Viscoelasticity Inside Living Cells Using an Active–Passive Calibrated Optical Trap. In: Gennerich, A. (eds) Optical Tweezers. Methods in Molecular Biology, vol 1486. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6421-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6421-5_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6419-2
Online ISBN: 978-1-4939-6421-5
eBook Packages: Springer Protocols