A simplified diagram of the optical layout of our instrument (see also ). The inset is a cartoon showing a detail of the specimen in the region illuminated by the three lasers (not to scale). Fluorescence excitation of the sample is supplied by evanescent wave illumination through the microscope objective over a region of several square micrometers (green). Overlapping within this region, the optical-trapping laser (pink) and position-detection laser (yellow) beams are coaxial and brought to diffraction-limited spots near the coverglass, as shown. An optically trapped bead tethered to the coverglass surface is shown for reference (blue). The area of regard of the fluorescence photodetectors through a confocal pinhole is indicated (dark gray). The main diagram shows the instrument itself, which is based on an inverted microscope with a nano-positionable three-dimensional piezo stage and equipped with a mercury arc lamp; the key components are shown in the center of the diagram. Also shown is the quadrant photodiode (QPD) subsystem used to detect changes in the position of the trapped bead. The input optics, including all three lasers, are shown to the right of the microscope inside the box labeled in red. The position-detection pathway is shown in orange, the trapping-laser pathway in red, the fluorescence-excitation pathway in blue and the fluorescence-emission pathway in dark green. The normal microscope transillumination pathway is shown in light green. The trapping laser beam can be moved electronically by means of acousto-optic deflectors (AODs) placed at optical planes conjugate to the back focal plane of the objective. The output optics, including a cooled, intensified charge-coupled device (CCD) camera, a conventional black-and-white CCD camera, and two silicon avalanche photodiodes (SAPDs), are shown to the left of the microscope, inside the box labeled in green. The identities of other optical elements are: B, beam; D, dichroic; F, filter; L, lens; P, polarizer; S, shutter; FM, flipper mirror.