Time-resolved single molecule fluorescence spectroscopy of Cy5-dCTP: influence of the immobilization strategy

The carbocyanine derivative Cy5 is one of the most utilized probes for single molecule fluorescence spectroscopy. To investigate time dependent changes in single molecules a number of immobilization techniques are used. In the present study the influence of the immobilization strategy on the fluorescence lifetime of Cy5-dCTP has been investigated using time-resolved single molecule imaging and spectroscopy techniques. The fluorescence lifetime of single dye molecule adsorbed on glass from aqueous solution (1.03 ns) or on a dry surface (1.23 ns) has been found to match very well with that measured in an ensemble aqueous solution (1.07 ns). However, the lifetime increases drastically when embedded in a film (approximately 2 ns). The changes in the rigidity of the medium have been found to be responsible for the unusual increase observed in the single molecule fluorescence lifetime of the dye. These observations were well supported by the results obtained during bulk measurements.