Multiparticle Tracking on a Multifocal Multiphoton Microscope (MMM)

We present a novel 3-D multiple-particle tracking technique that integrates a long focal length cylindrical lens into a multifocal multiphoton microscope (MMM). Due to the aberration induced by the cylindrical lens, the image projections of the fluorescent beads are circular in focus, and become elliptic above or below the focus. Calculating the width/ length ratio and the centroid of the ellipses in the MMM image, we can thus determine the z position and the x, y position of beads. Since the laser power is efficiently utilized in the MMM system, the acquisition time is greatly reduced, and enables the imaging of fast moving particles. Moreover, the video-based 3-D tracking technique is capable of multiple-particle tracking, which is especially valuable for gathering statistical information, and makes this system a promising tool in microrheology studies and other biological applications. We demonstrate the use of this technique to track multiple integrin-bound particles on fibroblast cells under mechanical stress.