Image analysis applied to luminescence microscopy

We have developed a novel approach to study luminescent light emission during migration of living cells by low-light imaging techniques. The equipment consists in an anti-vibration table with a hole for a direct output under the frame of an inverted microscope. The image is directly captured by an ultra low- light level photon-counting camera equipped with an image intensifier coupled by an optical fiber to a CCD sensor. This installation is dedicated to measure in a dynamic manner the effect of SF/HGF (Scatter Factor/Hepatocyte Growth Factor) both on activation of gene promoter elements and on cell motility. Epithelial cells were stably transfected with promoter elements containing Ets transcription factor-binding sites driving a luciferase reporter gene. Luminescent light emitted by individual cells was measured by image analysis. Images of luminescent spots were acquired with a high aperture objective and time exposure of 10 - 30 min in photon-counting mode. The sensitivity of the camera was adjusted to a high value which required the use of a segmentation algorithm dedicated to eliminate the background noise. Hence, image segmentation and treatments by mathematical morphology were particularly indicated in these experimental conditions. In order to estimate the orientation of cells during their migration, we used a dedicated skeleton algorithm applied to the oblong spots of variable intensities emitted by the cells. Kinetic changes of luminescent sources, distance and speed of migration were recorded and then correlated with cellular morphological changes for each spot. Our results highlight the usefulness of the mathematical morphology to quantify kinetic changes in luminescence microscopy.