Scanning FCS, a novel method for three-dimensional tracking

V. LEVI, Q. RUAN, K. KIS-PETIKOVA AND E. GRATTON1

BIOCHEMICAL SOCIETY TRANSACTIONS

Año: 2003 vol. 31 p. 997 - 1000

0300-5127

We describe a novel method to track fluorescent particles in three dimensions with nanometre precision andmillisecond time resolution. In this method, we use our two-photon excitation microscope. The galvomotordrivenx–y scanning mirrors allow the laser beam to move repetitively in a circular path with a radius ofhalf the width of the point spread function of the laser. When the fluorescent particle is located within thescanning radius of the laser, the precise position of the particle in the x–x plane can be determined by itsfluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective wasused to change the laser focus at two planes (half width of the point spread function apart). The difference ofthe fluorescence intensity in the two planes is used to calculate the z-position of the fluorescent particle. Thelaser beam is allowed to scan multiple circular orbits before it is moved to the other plane, thus improvingthe signal to noise ratio. With a fast feedback mechanism, the position of the laser beam is directed to thecentre of the fluorescent particle, thus allowing us to track a particle in three dimensions. In this contributionwe describe some calibration experiments performed to test the three-dimensional tracking capability ofour system over a large range.