| Abstract: | DRIMAPS (digitally recorded interference microscopy with automatic phase-shifting) is a system that we have developed for quantifying the behaviour of cells in subconfluent cultures. The primary data generated by the system consist of phase-shifted interference (PSI) images which are accurate density maps of the distribution of dry mass (non-aqueous material) inside cells. Time-lapse sequences of PSI images may be viewed as movie sequences or processed in various ways to reveal many different aspects of the dynamics of cell growth and motile behaviour. Here we describe the image processing routines that are an integral part of the system and are required for four main functions: 1 initial calculation of the PSI images; 2 compensation of these images for instrumental distortion and instability; 3 identification and tracking of individual cells in a time-lapse sequence of PSI images; 4 extraction of cell behavioural data from a time-lapse sequence of PSI images. The first function converts standard interference microscope images into an image that accurately represents the optical phase-difference introduced by the specimen. The second function recalibrates a sequence of images by taking the cell-free region in each image as a reference plane of zero phase-difference. This is particularly necessary to compensate for the long-term instability of the Horn type of double-beam interference microscope, which has several advantages over other types of interference microscope for studying cell behaviour. The third function compares consecutive images in a sequence in order to trace the identity of individual cells throughout the sequence. Semi-automatic tracking, which allows close interaction with a human operator, is less prone to error than fully automatic tracking. The fourth function automatically extracts dynamic data from the identified cells. These data may include the true mass centroids of cells for translocation analysis and robust morphometric parameters for cell morphology examination. The integrated intensity of a cell is an accurate measure of cell mass and allows the growth (increase in dry mass) of individual cells to be studied. These data may be entered into a relational database of cell behaviour and a rule-based system allows efficient data access and analysis. Experiments with phase-contrast microscopy have revealed that many of these image processing methods are generally useful for cell behaviour studies using more conventional forms of microscopy. |
