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

KETVALEE TREEGATE; AREEFEN RASAMESSARD; TANAKORN OSOTCHAN; JOSE HECTOR HODAK

SENSORS AND ACTUATORS B-CHEMICAL

ELSEVIER SCIENCE SA

Año: 2010 vol. 150 p. 239 - 246

0925-4005

This paper describes the construction of a positioning device for sample scanning in the x and y directionssuitable for single molecule fluorescence experiments. The mechanism uses a simple parallelogramflexure cut out of a single aluminum plate and two amplified piezoelectric actuators of the type usedfor microscope objective focus adjustment. A displacement range of 75 micrometers on each axis is obtained. Thestage can be used to implement a sample scanning confocal microscope for single molecule spectroscopyapplications using either inverted or up-right microscopes. Images with diffraction limited resolutioncan be obtained with this scanning stage. This is demonstrated by imaging glass beads labeled with theDY475 fluorescent dye and single rhodamine molecules. Micron sized range images of 256×256 pixelscan be obtained with dwell times down to 0.5 ms/pixel. A novel direct calibration in which the mechanicalresponse obtained from the line profiles for forward and reverse motion is used to account for the hysteresisof the stage. The target molecules are then located within the focus of the laser beam by using itscorrected position. The performance of this scanning device and correction technique are demonstratedfor the acquisition of fluorescence trajectories of individual rhodamine molecules.