In confocal and fluorescence microscopy, irradiation of the fluorophores with a focused laser beam at high power densities increases the emission intensity up to the point of dye saturation, a condition whose parameters are dictated by the excited state lifetime. In the excited state, fluorophores are unable to absorb another incident photon until they emit a lower-energy photon through the fluorescence process. When the rate of fluorophore excitation exceeds the rate of emission decay, the molecules become saturated and the ground state population decreases. As a result, a majority of the laser energy passes through the specimen undiminished and does not contribute to fluorophore excitation. Balancing fluorophore saturation with laser light intensity levels is, therefore, a critical condition for achieving the optimal signal-to-noise ratio in confocal experiments.

The adherent culture of male rat kidney kangaroo (PtK2) cells exhibited in the digital image above was immunofluorescently labeled with primary anti-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy2. In addition, the cells were stained with DAPI, which binds to DNA in the cell nucleus. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.