Research Article

Accurate calibration of optical tweezers close to a glass surface using interference rings in backscattered light

¹ Université Paris-Saclay, Institut d’Optique Graduate School, CNRS, Laboratoire Charles Fabry, 91127 Palaiseau, France
² Laboratoire d’Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau, France
³ Université Paris-Saclay, Ecole Normale Supérieure Paris-Saclay, CNRS, CentraleSupélec, LuMIn, 91190 Gif-sur-Yvette, France

^* Corresponding author: nathalie.westbrook@institutoptique.fr

Received: 13 February 2023
Accepted: 27 April 2023

Abstract

Mechanical forces play an important role in the behaviour of cells, from differentiation to migration and the development of diseases. Optical tweezers provide a quantitative tool to study these forces and must be combined with other tools, such as phase contrast and fluorescence microscopy. Detecting the retro-reflected trap beam is a convenient way to monitor the force applied by optical tweezers, while freeing top access to the sample. Accurate in situ calibration is required especially for single cells close to a surface where viscosity varies rapidly with height. Here, we take advantage of the well contrasted interference rings in the back focal plane of the objective to find the height of a trapped bead above a cover slip. We thus map the viscous drag dependence close to the surface and find agreement between four different measurement techniques for the trap stiffness down to 2 μm above the surface. Combining this detection scheme with phase contrast microscopy, we show that the phase ring in the back focal plane of the objective must be deported in a conjugate plane on the imaging path. This simplifies implementation of optical tweezers in combination with other techniques for biomechanical studies.