A series of fluorescein and rosamine derivatives have been prepared and their spectroscopical properties analyzed to determine their usefulness as donor and/or acceptors in "through-bond" energy transfer systems. Such new systems have been tailored to possess higher quantum yields, increased water solubility and higher pH independence. Some of these compounds have also been designed with handles for bioconjugation for use in intracellular imaging. The syntheses of most of these compounds have been optimized to afford higher yields in a multigram scale. Single molecule studies on a fluorescein/rosamine cassette are also reported.