Fluorescence phase-modulation fluorimetry

Two time-resolved fluorescence techniques, pulse Jluorimetry and phase-modulation fluorimetjy, are commonly employed to recover the lifetimes. The former uses a short exciting pulse (from femtoseconds to nanoseconds) of light, which leads to the pulsed response of the sample, which should then be deconvolved from the instrument response. In phase-modulation fluorimetry, the intensity of light used for excitation is modulated at a frequency whose reciprocal is similar to the fluorescence decay time. The sample response is also modulated, but with a time delay, measured as phase shift, from which the emission decay time can be calculated. Thus, the first technique works in the time domain, while the second one in the frequency domain. The most widely used technique in the time domain is the time-correlated single-photon counting [10, 11]. The merits of both techniques have been extensively discussed [12]. 

B) Phase-shift methods. The phase shift method for determining fluorescence lifetimes is based on the principle that if fluorescence is excited by suitably modulated light source, emitted radiation will also be similarly modulated. With reference to a scattering substance, emission from a fluorescent substance will introduce a time lag due to finite time between absorption and emission. This, by definition is the lifetime of the excited state. The time lag will cause a phase-shift relative to the exciting light. Phase fluorimetry requires a modulated light source and a phase sensitive detector. 

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