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 both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.
Maire
E
E
CNRS EP 560, Institut de Biologie de Lille/Institut Pasteur de Lille, France.
Lelièvre
E
E
Brau
D
D
Lyons
A
A
Woodward
M
M
Fafeur
V
V
Vandenbunder
B
B
D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D004285:Dogs; D007668:Kidney / Q000166:cytology / Q000201:enzymology; D008027:Light; D008156:Luciferases / Q000235:genetics; D008856:Microscopy, Fluorescence; D014158:Transcription, Genetic
D008156:Luciferases