Abstract
Publisher Summary This chapter describes frequency-domain fluorescent lifetime imaging microscopy (FLIM), which utilizes a sinusoidally modulated laser excitation source to excite the donor fluorophore. A frequency domain FLIM has a number of components. There are two main methods for measuring fluorescence lifetime, namely—time-domain and frequency-domain-based fluorescence lifetime sensing. The principles of frequency-domain-based fluorescence lifetime sensing, which is based on monitoring the extent of demodulation and phase shift between a sinusoidally modulated excitation light and the donor emission consequent on its excitation, are explained in this chapter. In time-domain FLIM, the sample is excited with a short pulse, and the subsequent fluorescence is integrated into separate time windows. The fluorescence lifetime is then calculated by fitting an exponential model to the measured intensities. This chapter focuses on the frequency-domain-based method, using a conventional inverted microscope configuration that employs phase-sensitive detection of fluorescence emission.
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