X–Y sample scanning stage and calibration method suitable for single-molecule detection

This paper describes the construction of a positioning device for sample scanning in the x and y directions suitable for single molecule fluorescence experiments. The mechanism uses a simple parallelogram flexure cut out of a single aluminum plate and two amplified piezoelectric actuators of the type used for microscope objective focus adjustment. A displacement range of 75 μm on each axis is obtained. The stage can be used to implement a sample scanning confocal microscope for single molecule spectroscopy applications using either inverted or up-right microscopes. Images with diffraction limited resolution can be obtained with this scanning stage. This is demonstrated by imaging glass beads labeled with the DY475 fluorescent dye and single rhodamine molecules. Micron sized range images of 256 × 256 pixels can be obtained with dwell times down to 0.5 ms/pixel. A novel direct calibration in which the mechanical response obtained from the line profiles for forward and reverse motion is used to account for the hysteresis of the stage. The target molecules are then located within the focus of the laser beam by using its corrected position. The performance of this scanning device and correction technique are demonstrated for the acquisition of fluorescence trajectories of individual rhodamine molecules.