Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells

We have developed an approach to study in single living epithelial cells bo th cell migration and transcriptional activation, which was evidenced by th e detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescen ce inverted microscope, connected to an ultralow-light-level photon-countin g camera and an image-acquisition card associated to specialized image anal ysis software running on a PC computer. Using a simple method based on a th in calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photo n-counting cameras designed to observe luminescence. This setup allows us t o measure by image analysis the luminescent light emitted by individual cel ls stably expressing a luciferase reporter vector. The sensitivity of the c amera was adjusted to a high value, which required the use of a segmentatio n algorithm to eliminate the background noise. Following mathematical morph ology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highl ight the usefulness of our image acquisition chain and mathematical morphol ogy software to quantify the kinetics of luminescence changes in migrating cells. (C) 2000 Academic Press.