pulsed or modulated at high frequency (up to 500 MHz) such as an LED, diode laser or a continuous wave source combined with an electro-optic modulator or an acousto-optic modulator. The fluorescence is (a.) demodulated and (b.) phase shifted; both quantities are related to the characteristic decay times of the fluorophore. Also, y-components to the excitation and fluorescence sine waves will be modulated, and lifetime can be determined from the modulation ratio of these y-components. Hence, 2 values for the lifetime can be determined from the phase-modulation method. The lifetimes are determined through a fitting procedures of these experimental parameters. An advantage of PMT-based or camera-based frequency domain FLIM is its fast lifetime image acquisition making it suitable for applications such as live cell research. Analysis The goal of the analysis algorithm is to extract the pure decay curve from the measured decay and to estimate the lifetime(s). The latter is usually accomplished by fitting single or multi exponential functions. A variety of methods have been developed to solve this problem. The most widely used technique is the least square iterative re-convolution which is based on the minimization of the weighted sum of the residuals. In this technique theoretical exponential decay curves are convoluted with the instrument response function, which is measured separately, and the best fit is found by iterative calculation of the residuals for different inputs until a minimum is found. For a set of observations of the fluorescence signal in time bin i, the lifetime estimation is carried out by minimization of: Besides experimental difficulties, including the wavelength dependent instrument response function, mathematical treatment of the iterative de-convolution problem is not straight forward and it is a slow process which in the early days of FLIM made it impractical for a pixel-by-pixel analysis. Non fitting methods are attractive because they offer a very fast solution to lifetime estimation. One of the major and straightforward techniques in this category is the rapid lifetime determination (RLD) method. RLD calculates the lifetimes and their amplitudes directly by dividing the decay curve into two parts of equal width t. The analysis is performed by integrating the decay curve in equal time intervals t: Ii is the recorded signal in the i-th channel and K is the number of channels. The lifetime can be estimated using: For multi exponential decays this equation provides the average lifetime. This method can be extended to