The QuantEM:512SC is an electron-multiplying (EM) CCD camera from Photometrics with 16-bit quantitative stability. It provides single-molecule sensitivity for ratiometric analysis in time-course experiments, reproducible data during long-term studies and streaming data capture for multidimensional time-lapse investigations. Advanced Clocking Enhancement technology and a field-programmable gate array enable self-calibrating gain linearization to 1000 times with a linear gain slider and a low generation rate of dark background events. An active regulation feedback system controls electron-multiplying gain and ensures the maintenance of factory-set parameters. Features include a back-illuminated frame-transfer EMCCD for >90% peak quantum efficiency and high-speed operation, and a Turbo 1394 FireWire interface for camera connectivity. The system may be employed for fluorescence recovery after photobleaching, total internal reflection fluorescence and fluorescence resonance energy transfer.