CCD imaging of luciferase gene expression in single mammalian cells

Abstract

Quantitative and sensitive imaging of chemiluminescence, bioluminescence and fluorescence emissions is emerging as an increasingly important technique for a range of biomedical applications (Hooper et al., 1990). A brief review of low-light-level imaging is presented, with particular reference to charge-coupled devices (CCD). Detectors for sensitive imaging are described and compared, including various CCDs and photoncounting devices. Image analysis techniques based on digital image processing, may be applied to quantify luminescent processes with these detectors. Images of luciferase gene expression in single mammalian cells have been obtained using a particular highsensitivity intensified CCD camera. The method is illustrated using cell monolayers infected with recombinant vaccinia virus encoding the firefly luciferase, luc gene (Rodriguez et al., 1988). The CCD camera has been used to detect luciferase expression in single, recombinant infected cells amongst over one million non-infected cells. The rapid detection of luciferase-expressing viruses may be used for the selection of virus deletion mutants into which the luciferase gene has been cloned at specific sites. This is particularly useful in the case of viruses such as cytomegalovirus which have slow replication cycles. This direct imaging technique is simple and versatile. It offers a rapid, non-invasive method for the sensitive detection of luciferase activity in single, luciferase-expressing cells. One can envisage the use of luciferase as a sensitive and convenient co-selection marker gene in the analysis of both gene expression and protein function. These methods offer tremendous potential in the fields of molecular and cellular biology.