Single Molecule Fluorescence and Optical Tweezers: Application to Molecular Motors

Alex E. Knight and Justin Molloy, Biology Department, University of York, YO1 5DD, UK

[Introduction] [Optical Tweezers] [TIRFM] [Problem] [Apparatus] [Experiment]

Introduction

Optical tweezers are a versatile tool for the manipulation of micron-sized objects. They use photon pressure from a tightly focused laser beam to ‘trap’ refractile particles. When combined with precise position sensors, they can be used to investigate mechanical interactions between individual protein molecules. In our lab, we measure forces and displacements in the piconewton and nanometre range, in order to understand the mechanism of the motor protein myosin. We are now combining this technique with single molecule fluorescence imaging to resolve the issue of coupling between the biochemical and mechanical events during the myosin ATPase cycle.

The Apparatus:

Our apparatus is built around a modified Zeiss Axiovert microscope. The optical tweezers are produced by a 3.3W diode-pumped infra-red laser. The beam is brought into the microscope directly underneath the objective, so that fluorescence filters can be changed without interrupting the beam. Two traps are generated by rapidly chopping the laser beam between two positions using acousto-optic deflectors, which also give independent control of the trap positions. The apparatus also incorporates a piezoelectric x-y microscope stage that has sub-nanometre position control.
To detect the binding and release of nucleotide by a single myosin head, it is necessary to use fluorescent ATP derivatives such as cy3 and cy5-ATP. These compounds have been shown to be useful substrates for myosin, and have exceptionally good fluorescence properties. We use Total Internal Reflection Fluorescence Microscopy (TIRFM), which gives very low background fluorescence, using either frequency-doubled Nd:YAG (532 nm) or HeNe (633 nm) laser light for excitation. Fluorescence is detected by a photomultiplier tube with a custom-built photon counting circuit, which gives a good signal to noise ratio and very high gain. The position sensors will use the trapping laser itself, rather than bright field illumination, to monitor the position of the bead, as this is more compatible with simultaneous low-level fluorescence measurements.
Data acquisition and system control are achieved by computer, using a custom interface board and software. This permits us to collect data at the high sampling rates required to resolve individual force generating and fluorescence events.