Three-dimensional image acquisition system for multi-sperm tracking

Sperm motility has been widely studied in two dimensions (2D) by analyzing their bidimensional trajectories when swimming near a flat surface. Under real conditions, the spermatozoid swims in a three-dimensional space before finding its target, the egg. The main restriction to track three-dimensionally these flagellated cells is their speed. Here we describe a novel method that allows the acquisition of data for three dimensional multi-tracking and analysis of free-swimming sperm trajectories. The system uses a piezoelectric device displacing a large focal-distance objective mounted on an inverted microscope (over its optical axis) to acquire 70 stacks of 60 images at a rate of 4,200 images of 512 times 512 pixels per second, over a depth of 100 mum. With this method whole 3D paths could be visualized during 1 s and measured for multiple sperm in the same field simultaneously for the first time. This new acquisition method extends the analytic possibilities from a restricted 2D situation to real 3D conditions.