Live-cell fluorescence imaging.

This chapter examines the ways to optimize the signal-to-noise ratio while keeping the specimen healthy. Live cells expressing fluorescent protein fusions are usually dim compared to fixed specimens, both because the fluorescent proteins are not very bright and because there is, in most cases, only one fluorophores per protein. It is also favorable to choose cells that are expressing low levels of fluorescent protein fusions to minimize the difference from the levels of the endogenous protein in vivo. Long camera exposure times, which allow accumulation of weak signals, must be often avoided to reduce photobleaching and phototoxicity and to acquire images quickly enough to capture cell dynamics. Choices, such as objective lens and camera, determine the signal-to-noise ratio of an imaging system. Optimizing the imaging system to maximize signal and minimize noise is critical for live-cell fluorescence imaging. Imaging with high signal-to-noise ratio will allow detection of low concentrations of fluorescent fusion proteins with illumination conditions that are less likely to damage cells. Automation of an imaging system allows collection of multidimensional data while helping to maintain focus and minimize specimen exposure to light. Under all imaging conditions, maintaining and verifying cell health is essential to the validity of the experimental results.