A Minimal Optical Trapping and Imaging System

Abstract

We have constructed and tested a simple and versatile optical tweezers apparatus made from individual optical components, capable of visualizing individual microtubules (∼25 nm in diameter) and studying biomolecules at the single-molecule level, using a minimal set of parts. Our design is based on a conventional, inverted microscope operating under plain bright field illumination. The illuminating system of the microscope is composed of a current-stabilized LED source, a collector lens system, a field diaphragm, a condenser iris diaphragm, a relay lens (LR) that images the condenser iris diaphragm onto the condenser back focal plane (BFP), and the condenser. All of these elements are adjusted for Koehler illumination. On the other hand, a single IR laser beam, coupled into the microscope, enables both standard optical trapping and BFP detection for the measurement of molecular displacements and forces. A convenient feature of our optical setup is that the relay lens LR effectively decouples the illuminating system from the optical tweezers detection stage. In practice, we can close the condenser iris diaphragm almost entirely (∼90%) to maximize contrast for sample visualization, while simultaneously using the full numerical aperture of the condenser for BFP detection. We are able to further optimize imaging of the microscope by using computer-based digital image processing, effectively removing the background, reducing noise, and enhancing contrast. Altogether, our system yields excellent sample visualization in real-time, without the use of any specialized optics and without affecting the optical tweezers optical path. We have tested the optical trapping instrument by measuring the persistence length of double-stranded DNA at high ionic strength (150 mM of Na+), and by following the stepping of the motor protein kinesin on clearly imaged microtubules. The approach presented here provides a straightforward alternative for the study of biomaterials and individual biomolecules.