These fluorescent microscopic techniques were based on intensity measurements of live cells, tissues, and organs in which there is a significant degree of tissue heterogeneity, light scattering, and a multitude of physical factors and conditions that affect the intensity measurements. The implementation of fluorescent lifetime measurements and imaging in the field of biomedicine is an attempt to overcome the inherent difficulties of intensity measurements in absolute units. This alternative methodology is based on the assumption that within the duration of the fluorescence decay, the fluorescence lifetime measurements are independent of the fluorophore concentration, the instrumentation for the excitation, and the detection instrumentation of the fluorescence decay signal. The modern advances are due to several factors: ultrafast lasers and light sources, advances in analog and digital signal processing, advances in detector design, and new analytical methods to analyze the decay data, and the ability to form images that are based on the decay data. The new instrumentation and analytical approaches are still dependent of the numbers of detected photons and the signal-to-noise ratios, but they are useful for live cell studies and human clinical studies when they are validated with appropriate control measurements. The new advances in both instrumentation and data analysis techniques that are comprehensively and clearly elucidated in this book permit the application of these methods to investigations of live cells and to in vivo studies.