The ability to detect and even to identify a single fluorescent dye via its characteristic fluorescence properties opens up a wide range of new opportunities for ultrasensitive analytical applications in chemistry, biology, and medicine. Previously, we have only been using the fluorescence lifetime of a dye for its identification and quantification at one- [1,3,7,10] and coherent two-photon excitation [2,4,5,6]. This ability makes it possible to study conformational dynamics of DNA by monitoring changes in the fluorescence lifetime and intensity . Additional properties of a fluorescing molecule are its fluorescence anisotropy, intensity, and emission spectrum. Extracting all of these fluorescence information significantly increases the sensitivity of single molecule fluorescence detection for diagnostic applications, such as monitoring enzyme function, rare event detection, probe-target binding, and conformational dynamics of biomolecules. Therefore, we are employing a confocal epi-illuminated fluorescence microscope, a polarizing beam-splitter in conjunction with dual channel detection, and the real-time spectroscopic technique BIFL (BIFL records two different information on each signal photon: the arrival time relative to the exciting laser, as well as the time lag to the preceding photon [7,10,13]) (set-up), to measure and monitor the fluorescence lifetime, intensity, and anisotropy during a single-molecule transit through the microscopic, open detection volume. We are able to identify and to quantify different single dye molecules in aqueous solution via their anisotropy . Furthermore, we are working on applications of the multidimensional confocal fluorescence spectroscopy together with fluorescence correlation spectroscopy (FCS) to the study of conformational dynamics of DNA and of biological processes [9,12].
To strive for an ultimate statistical accuracy in such single-molecule experiments, as many fluorescence photons as possible must be detected during a single molecule transit. However, increasing the excitation irradiance is limited by fluorescence saturation and photobleaching of the dye and a concomittant decrease of the signal-to-background ratio for one- [6,8,11] and coherent two-photon excitation [2,5,6]). Therefore, it is crucial to choose a dye with appropriate fluorescence properties, such as a high absorption coefficient, high fluorescence, low triplet, and low photobleaching quantum yield . We are using our confocal microscopy set-up together with fluorescence correlation spectroscopy (FCS) to investigate and discuss the effect of triplet population and photobleaching on the suitability of dye for SMD in solution using one-(OPE) or coherent two-photon excitation (TPE). For OPE of, e.g., Rhodamines, a considerable increase of the probability of photobleaching is observed at irradiances above 1 kW/cm2, which are commonly used in single-molecule experiments. Such an increase can only be described by photobleaching reactions from higher electronic excited states [6,8,11]. For Coumarins, this increase is even more severe by the use of pulsed excitation . We are investigating added compounds, that reveal strong stabilization properties especially at high irradiances  or strong triplet quenching properties, and, thus, cause a significant increase in the count rate per molecule of single-molecule experiments. These photophysical and -chemical aspects are discussed with regard to the signal-to-background ratio and the burstsize distribution (number of detected photons per single molecule transit ) of a single-molecule experiment in solution, to obtain optimal experimental conditions with respect to the excitation irradiance and the focal diameter [8,11].
 Börsch M., et al., Ital. Biochem. Soc. Trans. 11, 47 (1998)