Abstract
Holographic optical tweezers (HOTs) enable the manipulation of multiple traps independently in three dimensions in real time. Application of this technique to force measurements requires calibration of trap stiffness and its position dependence. Here, we determine the trap stiffness of HOTs as they are steered in two dimensions. To do this, we trap a single particle in a multiple-trap configuration and analyze the power spectrum of the laser deflection on a position-sensitive photodiode. With this method, the relative trap strengths can be determined independent of exact particle size, and high stiffnesses can be probed because of the high bandwidth of the photodiode. We find a trap stiffness for each of three HOT traps of kappa approximately 26 pN/microm per 100 mW of laser power. Importantly, we find that this stiffness remains constant within +/- 4% over 20 microm displacements of a trap. We also investigate the minimum step size achievable when steering a trap with HOTs, and find that traps can be stepped and detected within approximately 2 nm in our instrument, although there is an underlying position modulation of the traps of comparable scale that arises from SLM addressing. The independence of trap stiffness on steering angle over wide ranges and the nanometer positioning accuracy of HOTs demonstrate the applicability of this technique to quantitative study of force response of extended biomaterials such as cells or elastomeric protein networks.
Highlights
In recent years, optical tweezers [1] have emerged as a leading technique in the field of biophysics, due to their ability to constrain the position of micrometer-sized objects in three dimensions and to detect and exert biologically relevant picoNewton forces
For each change in the position of one or more of the Holographic optical tweezers (HOTs) traps, the entire kinoform sent to the spatial light modulator (SLM) is changed
To perform quantitative force measurements using optical traps, parameters such as trap stiffness and its position dependence, range of trap steering, minimum step size and trap stability are of key importance
Summary
Optical tweezers [1] have emerged as a leading technique in the field of biophysics, due to their ability to constrain the position of micrometer-sized objects in three dimensions and to detect and exert biologically relevant picoNewton forces. Extending the manipulation and force measurement capabilities of optical tweezers to the mechanical probing of higher-order biological structures, such as cells and soft biomaterials, requires the ability to stretch these higher-dimensional structures in more than one direction. This has been accomplished for example using ‘time-sharing’ to trap multiple microspheres bound to a red blood cell [5]. The quantitative interpretation of measured forces relies on the ability to scan the laser among locations much faster than the characteristic response time of the trapped object + material [6, 8]; a time that is not known a priori, and which, especially for stiffer biomaterials, may be significantly faster than accessible steering speeds
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