With the development of advanced ultrasensitive fluorescence detection methods, it is possible to perform experiments on single biomolecules and particles. In single molecule experiments, molecules or complexes are typically either immobilized on a surface or snap-shots of individual molecules are collected as they diffuse through the diffraction-limited observation volume of a confocal microscope. A third variation, single particle tracking, allows measurements on individual particles over time scales longer than a few milliseconds. Particularly in the cellular environment, a myriad of information can be gained by following individual biomolecules as they perform their function. Single particle tracking has been used to investigate the movement of lipids in the plasma membrane [1
], follow individual viruses along their infection pathway [4
] or study the motion of individual complexes within living cells (e.g. [7
There has been a large increase during the last years in the development of 2-dimensional and 3-dimensional single-particle tracking methods. In general, two approaches are followed for single-particle tracking: either tracking is performed ex post facto
from data collected with a camera via post-processing or the tracking is performed in real-time using a feedback approach. The former approach has the advantage that multiple particles can be tracked simultaneously whereas the latter approach is typically faster and more flexible. An elegant variation of the second approach was developed by Cohen and Moerner who used a feedback loop to cancel Brownian motion by electrokinetic forces induced by varying the voltage on electrodes near the particle and thereby trapping the particle [9
]. However, this approach is not currently applicable to measurements in living cells.
Two similar methods for 3-D particle tracking using feedback have been realized: tetrahedral tracking by Berg [10
] and Werner [11
] and orbital tracking by Gratton [12
]. In the groups of Berg and Werner, four detectors measure different volumes arranged in a tetrahedral geometry about the focus of a confocal microscope and are used to detect motion of a particle away from the focus. The sample stage is then moved to place the tracked particle back into the center of the confocal volume. In orbital tracking, the laser is orbited around the tracked particle. Detection of the z
-position is performed in two planes, one above and a second below the tracked particle. A feedback algorithm is used to keep the orbit centered on the particle being tracked.
One of the major criticisms of the orbital tracking method is that one is blind to the local environment. Here, we present a modified version of 3-D orbital tracking where wide-field images can be simultaneously collected. Hence, we are able to probe the local environment of the tracked particle while maintaining the advantages of the feedback tracking method. The trajectories determined from tracking individual particles can be used to provide a nanoscopic view of the particles behavior within the context of the living cells. From the trajectories of single particles, the maximum amount of information available regarding the motional behavior of the tracked particle can be gained including the diffusion coefficient, instantaneous and average transport velocities, or corralled radius [14
]. As the motion within cells is anisotropic, 2D tracking can lead to artifacts and misleading results. With the full, 3D information, the anisotropies can be investigated in detail. In addition, simultaneous wide-field imaging allows visualization of the cellular environment about the individual particles and the interaction of the tracked particles with different cellular components.