A minimal optical trapping and imaging microscopy system.

Carmen Noemí Hernández Candia,Sara Tafoya Martínez,Braulio Gutiérrez-Medina

doi:10.1371/journal.pone.0057383

Carmen Noemí Hernández Candia, Sara Tafoya Martínez + Show 1 more

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https://doi.org/10.1371/journal.pone.0057383

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Abstract

We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter) and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

Highlights

Over the last two decades, optical traps have become a standard tool in the physical and biological sciences, allowing the measurement of sub-nanometer displacements of optically trapped microparticles, as well as the exertion of piconewton-level, controlled forces on these particles [1,2,3]
An optical tweezers arrangement consists of a single laser beam tightly focused by a microscope objective of high numerical aperture (NA) (1.0–1.4), which traps microparticles near its focal point [5,6]
The end terminal of the laser fiber is secured onto the optical table, and a combination of a half-wave plate and a polarizing cube beamsplitter (PBS) control the laser power going to the optical trap

Summary

Introduction

Over the last two decades, optical traps (or optical tweezers) have become a standard tool in the physical and biological sciences, allowing the measurement of sub-nanometer displacements of optically trapped microparticles, as well as the exertion of piconewton-level, controlled forces on these particles [1,2,3] Taking advantage of this versatility, studies on the mechanical and biochemical properties of biomolecules at the single-molecule level using optical tweezers are widespread. In these assays, individual molecules, attached to trapped particles, can be pulled on or stretched (or even twisted) using the laser trap, and the molecular displacement and force (or torque) responses can be measured with high spatial (,1 nm) and temporal (,100 kHz) resolution [4]. This last option is the one we have followed in this work

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