A quantitative fluorometer for multiple fluorophores having a separate time-modulated beam of excitation light for each fluorescent target (26). Each beam is synchronized with one or more detectors (114 and 116) and separate lock-in amplifiers (30 and 32). The fluorophores are simultaneously excited and the combined fluorescent emission is resolved into components corresponding to each fluorophore. Confocal scanning means (24) are used to excite and detect fluorescent emission from locations throughout a volume. The location specific output of each amplifier is stored in a computer (34) which resolves the emission into the components corresponding to each fluorophore. The location specific data may be further processed or visually displayed to provide simultaneously fluorescence lifetime images of two fluorophores with this frequency domain method. Multiple amplifiers for each detector channel allow phase discrimination in each channel so that prompt and delayed fluorescence may be measured, allowing use of multiple fluorophores in each detector channel.
APPARATUS FOR QUANTITATIVE IMAGING OF MULTIPLE FLUOROPHORES