Confocal and force probe imaging system for simultaneous three-dimensional optical and mechanical spectroscopic evaluation of biological samples

Abstract

We present the design and operation of a novel instrument for the simultaneous three-dimensional measurements of localized properties using optical and mechanical probes. In this instrument the mechanical and optical probes are stationary relative to the instrument frame while the specimen can be navigated in three-dimensional space in the probing field, translating over a range of 64.5 microm by 49.7 microm by 31.5 microm in each axis, respectively, at closed loop speeds of 10 Hz. A large aperture is provided in the center of the moving platform so that an optical lens can image the specimen from below. An additional z-direction translator has been integrated with this instrument to independently move a force probe that contacts the specimen from above with a translation range of 16 microm. Furthermore, there is an additional seven degrees of freedom providing adjustments to independently position and/or align the scanner and force probe relative to the optical imaging lens. Initial results of both optical and mechanical scans demonstrate 6 nm localization from single molecule fluorescence measurements, as well as single pair fluorescence energy transfer measurements indicating molecular separations of about 2 nm.