Current Pharmaceutical Biotechnology, 2009, 10, 467-473 467 1389-2010/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Single-Molecule Force Spectroscopy Using the NanoTracker™ Optical Tweezers Platform: from Design to Application A. Wozniak a, *, J. van Mameren a and S. Ragona b a JPK Instruments AG, Bouchéstrasse 12, 12435 Berlin, Germany; b Sid Ragona, Ragona Scientific, Pittsford, NY 14534, USA Abstract: Since the development of detection and analysis techniques for optical tweezers setups, there has been an ever- increasing interest in optical tweezers as a quantitative method, shifting its applications from a pure manipulation tool to- wards the investigation of motions and forces. With the capability of manipulation and detection of forces of a few hun- dred picoNewtons down to a fraction of a picoNewton, optical tweezers are perfectly suitable for the investigation of sin- gle molecules. Accordingly, the technique has been extensively used for the biophysical characterization of biomolecules, ranging from the mechanical and elastic properties of biological polymers to the dynamics associated with enzymatic ac- tivity and protein motility. Here, the use of state-of-the-art optical tweezers on the elasticity of single DNA molecules is presented, highlighting the possibilities this technique offers for the investigation of protein-DNA interaction, but also for other single molecule applications. Technical in nature, design aspects of the NanoTracker™ optical tweezers setup are addressed, presenting the recent advances in the development of optical tweezers, ranging from noise reduction to detec- tion and calibration methodology. Keywords: Optical tweezers, dual beam, force measurements, power spectrum calibration, quadrant photodiode, back focal plane interferometry, DNA elasticity, hysteresis. INTRODUCTION Optical tweezers are a non-invasive technique to trap, manipulate and track particles. The basic physical principle underlying optical tweezers is the radiation pressure exerted by light when colliding with matter. The effect was postu- lated and first demonstrated by Arthur Ashkin in the late 1960s. It has become known as optical trapping, in which dielectric particles can be stably trapped in three dimensions in a highly focused laser beam. Since the advent of optical tweezers, the technique has developed tremendously, shifting its application from physi- cal to biological sciences. A pure manipulation tool at the beginning, it has been – and still is – used for particle sorting [1] and microfabrication [2]. As cells and organelles can be trapped directly thanks to their optical density, they were the first to come into the ‘focus’ of biological optical tweezers applications. Optical tweezers were used to sort cells and test the viscoelastic properties of the cellular cytoskeleton. With the development of detection and analysis techniques, optical tweezers have found their way into biochemical and bio- physical sciences. It is now widely used as a quantitative tool to exert calibrated forces on microscopic objects and simul- taneously measure the displacements and forces generated by these systems. In order to measure the forces associated with single molecule interactions and kinetics, the biomolecule of interest is often attached to a probe. The optical tweezers probe – a bead with the diameter of several tens of nanome- ters to several micrometers – can be used to perform force *Address correspondence to this author at the JPK Instruments AG, Bou- chéstrasse 12, 12435 Berlin, Germany; Tel: +49 30 5331 12070; Fax: +49 30 5331 22555; E-mail: wozniak@jpk.com; nanotracker@jpk.com spectroscopy, tracking or imaging experiments. Owing to optical tweezers, a lot could be learned about the mechanics of molecular motors. Stepping of kinesin and dynein on mi- crotubule filaments [3,4], base-pair stepping of RNA polym- erase [5] as well as codon stepping of ribosomes [6] were detected. A significant number of reports were also pub- lished on stretching and the elasticity of DNA [7-10], of which the follow-up experiments are presented in this report. Furthermore, the technique has been successfully used by researchers in the field of microrheology [11], and the detec- tion of diffusion in cell membranes represents the ground- breaking experiments of quantitative optical tweezers on cells [12]. The detection of single-molecule forces and fast bio- chemical processes with high resolution relies on multiple parameters that have to be considered when setting up the optical tweezers system and the experimental conditions. In this report, we present the first force measurements on DNA performed on an optical tweezers setup living up to the latest standards in this field, the JPK NanoTracker™ platform. PRINCIPLE OF FORCE MEASUREMENTS The traditional way of macroscopic force measurement is the use of a Hookean spring, whose extension is proportional to the force acting on it. A system consisting of a laser focus with an intensity gradient and a spherical particle with a re- fractive index higher than the surrounding acts just like such a Hookean spring. In the absence of any external force acting on the particle, the focused laser light pushes the trapped particle into the center of the trap. This gradient force is the actual principle behind manipulation of objects with optical tweezers. Brownian motion, viscous drag or any other force