Fluorescence microscopy is a well-established platform for biology and biomedical research (Chapter 2). Based on this platform, fluorescence lifetime imaging microscopy (FLIM) has been developed to measure fluorescence lifetimes, which are independent of fluorophore concentration and excitation intensity and offer more information about the physical and chemical environment of the fluorophore (Chapter 3). The frequency domain FLIM technique offers fast acquisition times required for dynamic processes at the sub-cellular level. A conventional frequency-domain FLIM system employs a CCD camera and an image intensifier, the gain of which is modulated at the same frequency as the light source with a controlled phase shift (time delay). At the moment these systems, based on modulated image intensifiers, have disadvantages such as high cost, low image quality (distortions, low resolution), low quantum efficiency, prone to damage by overexposure, and require high voltage sources and RF amplifiers. These disadvantages complicate the visualization of small sub-cellular organelles that could provide valuable fundamental information concerning several human diseases (Chapter 3 and 4). In order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems, we have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM) (Chapter 5). The power of the light source needed for illumination in a FLIM system and the signalto-noise ratio (SNR) of the detector have led us to a photon “budget”. A light source of only a few milliWatts is sufficient for a FLIM system using fluorescein as an example. For every 100 photons emitted, around one photon will be converted to a photoelectron, leading to an estimate for the ideal SNR for one fluorescein molecule in an image as 5 (14 dB). We have performed experiments to validate the parameters and assumptions used in the mathematical model. The transmission efficiencies of the lenses, filters, and mirrors in the optical chain can be treated as constant parameters. The Beer-Lambert law is applicable to obtain the absorption factor in the mathematical model. The Poisson distribution assumption used in deducing the SNR is also valid. We have built compact FLIM systems based on new designs of CCD image sensors that can be modulated at the pixel level. Two different designs: the horizontal toggled MEM-FLIM1 camera and vertical toggled MEM-FLIM2 camera are introduced (Chapter 6). By using the camera evaluation techniques described in Chapter 7, these two versions of the MEM-FLIM systems are extensively studied and compared to the conventional image intensifier based FLIM system (Chapter 8). The low vertical charge transport efficiency limited the MEM-FLIM1 camera to perform lifetime experiments, however, the MEM-FLIM2 camera is a success. The MEM-FLIM2 camera not only gives comparable lifetime results with the reference intensifier based camera, but also shows a much better image quality and reveals more detailed structures in the biological samples. The novel MEM-FLIM systems are able to shorten the acquisition time since they allows recording of two phase images at once. The MEM-FLIM2 camera is, however, not perfect. It can only be modulated at a single frequency (25 MHz) and requires that the light source be switched off during readout due to an aluminum mask that had a smaller area than intended. A redesign of the architecture based on the vertical toggling concept leads to the MEM-FLIM3 camera (Chapter 9). Several improvements have been made in the sensor design for the MEMFLIM3 camera, such as higher fill factor, greater number of pixels etc. The MEM-FLIM3 camera is able to operate at higher frequencies (40, 60 and 80 MHz) and has an option for electron multiplication. Evaluations of this updated MEM-FLIM system are presented (Chapter 10). The images obtained from the MEM-FLIM3 camera at 20 and 40 MHz can be used directly for the lifetime calculation and the obtained lifetimes are comparable with the ones from the reference camera. There are, however, differences in the even and odd columns (20 MHz) and four image sections (40 MHz) for the intensity and lifetime images. For higher frequencies (60 and 80 MHz) calibrations are needed before calculating lifetimes. The lifetimes derived from the modulation depth after the calibrations are in a reasonable range while the lifetime derived from the phase cannot be used. At 60 and 80 MHz we can use one phase register from the MEM-FLIM3 camera for the lifetime calculation, the same way the reference camera operates. The lifetimes obtained by this method from the MEM-FLIM3 at 60 and 80 MHz are comparable with the ones from the reference camera. The MEM-FLIM3 camera also has an electron multiplication feature for low-light experimental condition. We could get approximately 500 times multiplication. Lifetime measurement using the EM function, however, has not been tested due to the limitation of the project time.