Fluorescence Lifetime Imaging System using a double-modulated Excitation Source

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Fluorescence lifetime imaging systems based on heterodyne detection using two dimensional photo-detector and a double-modulated excitation source have been developed. An image intensifier (I.I., Model V3063U, Hamamatsu Photonics Co.) was used as the two dimensional photo-detector.The gain control characteristics of the photo-cathode in the I.I. Tube are utilized to allow heterodyne detection with low-voltage control signal. Heterodyning the modulated fluorescence and reference signals at the photo-cathode of the I.I. Tube provides high-frequency response up to a few hundred MHz.In order to detect DC fluorescence image and fluorescence lifetime information simultaneously, Ar ion laser beam intensity as an excitation source was modulated at different frequencies, simultaneously, by tandem A/O modulators. One of the A/O modulators was modulated at a difference frequency between the modulation frequency of 20,000MHz for the other A/O modulator and the gain modulation frequency of 19,990MHz for the I.I. tube.A CCD camera coupled to the I.I. Measures the heterodyned fluorescence image and stores them in the memory of a personal computer. The phase resolved image, I.e. the life time image is calculated the stored data. The time resolution of the systems is about 1 ns.