Fluorescent dyes for multiphoton bio-imaging applications

Fluorescent dyes and probes are key components in multiphoton based fluorescence microscopy imaging of biological samples. While many commercially available fluorescent dyes have sufficed, most exhibit relatively low two-photon absorption (2PA) cross-section values in the tunability range of Tirsapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with either low fluorescence quantum yields and/or the additional problem of rapid photobleaching upon exposure to the high peak powers provided by fs laser sources. In order to address the demand for better performing dyes for two-photon based imaging, we have prepared a new series of reactive fluorophores tailored for multiphoton imaging. These fluorophores are based upon the fluorene ring system, known to exhibit high fluorescence quantum yields, typically > 0.7, and possess high photostability. They have been functionalized with moieties to act, e.g., as efficient amine-reactive fluorescent probes for the covalent attachment onto, e.g., proteins and antibodies. The synthesis and the single-photon spectral characteristics, as well as measured two-photon absorption cross sections of the reactive fluorophores in solution will be presented. Spectral characterizations of bovine serum albumin (BSA) conjugated with the new reactive probe will also be presented.