Analysis of moving biological objects in video microscopy sequences

We present a number of methods to detect and track multiple moving biological objects in image sequences acquired by different imaging techniques coupled to video microscopy. Movement and motility analysis is an important topic in biology and it is of major importance to be able to analyze the image sequences in order to get reliable and reproducible quantitative data such as number, position, movement phases and speed of the biological objects, as this information helps to characterize the biological assays. The detection is automatic and, in the case of phase contrast microscopy, is based upon the correlation of the image with a filter which varies adaptively to represent an object as it moves and deforms; in fluorescent imaging, the automatic detection is based on thresholding and mathematical morphology to determine and select the objects. The tracking is performed using a Kalman filter and a cost function which enable the position of the moving objects to be predicted, refined and updated. Once all moving objects have been assigned with unique spatio-temporal paths, trajectories are analyzed in terms of different parameters relevant for the motion analysis of biological objects.