NANO LETTERS
Three-Dimensional Particle Tracking via Bifocal Imaging
2007 Vol. 7, No. 7 2043-2045
Erdal Toprak,†,§ Hamza Balci,‡,§ Benjamin H. Blehm,‡ and Paul R. Selvin*,†,‡ Department of Biophysics, UniVersity of Illinois UrbanasChampaign, Urbana, Illinois 61801, and Department of Physics, UniVersity of Illinois UrbanasChampaign, Urbana, Illinois 61801 Received April 18, 2007; Revised Manuscript Received June 6, 2007
ABSTRACT We introduce a bifocal imaging method that enables three-dimensional (3D) tracking of both fluorescent and nonfluorescent particles. We accomplish this by simultaneously imaging a focused plane, for in-plane position (x,y), and a defocused plane, for out-of-plane position (z), of a molecule using a single CCD camera. We applied our method to several systems including in vivo melanosome tracking and phagocytosed fluorescent bead tracking. We have achieved 2−5 nm accuracy with a 2−50 ms time resolution.
Three-dimensional (3D) particle tracking has long been a challenge in various fields of science ranging from fluid mechanics to biology. On the other hand, the two-dimensional imaging field is significantly more mature: fluorescence imaging with one nanometer accuracy (FIONA), is able to track individual fluorophores with 1.5 nm accuracy;1 single molecule high-resolution colocalization (SHREC)2 and photoactivated localization microscopy (PALM)3 are able to get
