3-D Particle Tracking in a Two-Photon Microscope: Application to the

VALERIA LEVI, QIAOQIAO RUAN, AND ENRICO GRATTON

BIOPHYSICAL JOURNAL

Año: 2005 vol. 88 p. 1 - 10

0006-3495

ABSTRACT We developed a method for tracking particles in three dimensions designed for a two-photon microscope, whichholds great promise to study cellular processes because of low photodamage, efficient background rejection, and improveddepth discrimination. During a standard cycle of the tracking routine (32 ms), the laser beam traces four circular orbitssurrounding the particle in two z planes above and below the particle. The radius of the orbits is half of the x,y-width of the pointspread function, and the distance between the z planes is the z-width of the point spread function. The z-position is adjusted bymoving the objective with a piezoelectric-nanopositioner. The particle position is calculated on the fly from the intensity profileobtained during the cycle, and these coordinates are used to set the scanning center for the next cycle. Applying this method,we were able to follow the motion of 500-nm diameter fluorescent polystyrene microspheres moved by a nanometric stage ineither steps of 20–100 nm or sine waves of 0.1–10 mm amplitude with 20 nm precision. We also measured the diffusioncoefficient of fluorospheres in glycerol solutions and recovered the values expected according to the Stokes-Einsteinrelationship for viscosities higher than 3.7 cP. The feasibility of this method for live cell measurements is demonstrated studyingthe phagocytosis of protein-coated fluorospheres by fibroblasts.