Two-photon interactions at single fluorescent molecule level

Single-molecule spectroscopy and single-molecule detec-tion are emerging areas that have many applications when combinedwith scanning, imaging, and spectroscopy techniques. We have com-bined a commercial confocal scanning head, to a Ti:sapphire laserand to an inverted microscope, for the detection of single moleculefluorescence of varies dyes by two-photon excitation. We collectedspot images of fluorescent molecules that have been deposited on asubstrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interac-tions that occur with the following fluorescent molecules: Indo-1,Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specificdyes is based upon their wide use in biological and medical applica-tions together with the varying complexity of their chemical structurethat increases from Pyrene to Indo-1. Moreover, we report some dataabout single molecule studies related to denaturation of an enhancedgreen fluorescent protein, GFPmut2, under one photon excitation re-gime, that show a very similar trend to that observed for the alreadymentioned fluorescent molecules