Chapter 2 Frequency domain FLIM theory, instrumentation, and data analysis

Publisher Summary This chapter gives an overview on the governing equations of frequency domain lifetime imaging—specifically, apparent lifetimes, fluorophore mixtures, chi-squared minimization, discrete Fourier processing, and treatment of binary mixtures. In the frequency domain, rapid and minimally intrusive measurement involves a set of tradeoffs between lifetime accuracy and speed. The chapter focuses on instrumentation variations and trends in the field. The chapter presents data, primarily from wide field frequency domain measurements; however, wide ranges of confocal solutions in the frequency domain have been reported, including: point scanning, programmable arrays, and spinning disks. The principles of frequency domain lifetime imaging in both wide field and confocal measurements are the same. The implementation of all types is very similar with the exception of the point scanning reference, which uses a lock-in technique. The chapter presents the collection and processing of frequency domain data, and briefly describes subsequent processing of single or multiple lifetime images to provide information to users.