Coincident time-shared single molecule imaging, manipulation and bright-field microscopy
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An apparatus that combines single molecule fluorescence, optical trapping and bright-field microscopy is presented. Given the spread over orders of magnitude of the light intensities for the different techniques, special considerations in choosing the spectral regions for each were taken. Moreover, imaging single molecules in a background of intense light from the infra red laser used for the optical trap has been shown to result in enhanced photo-bleaching due to two-photon processes. A scheme for fast time-sharing was implemented in which the fluorescence excitation light and the trap light alternate in fast succession. This eliminates two-photon effects and enables stable manipulation using the optical trap. The simultaneous use of the bright-field imaging in differential interference contrast arrangement enables observation of refractile objects in the sample over large distances. The apparatus was designed for future use in studies of molecular motor regulation. However, to demonstrate the functionality of the system, the rotational diffusion of a micro-sphere fluorescently labelled at one spot was measured.