Multiparametric Detection of Fluorescence Emitted from Individual Multichromophoric Systems

While few years ago the main goal in room temperature single molecule fluorescence spectroscopy (SMS) was to visualize individual molecules, nowadays experiments are designed such that multiparametric observation of different fluorescence characteristics of the investigated molecular systems is allowed. The simultaneous observation of fluorescence characteristics such as spectral peak position, fluorescence decay times, polarization properties or wavelength integrated intensity of fluorescence during the survival time of the investigated molecules leads to a more detailed picture of the molecular states and environment changes experienced by the probed molecules. In this contribution a diffraction-limited scanning stage confocal microscope set-up allowing real time multiparametric observation of fluorescence detected from single molecules immobilized in thin polymer matrix is described. By using pulsed excitation in combination with burst integrated fluorescence lifetime (BIFL) type detection, the simultaneous acquisition of fluorescence spectra, wavelength integrated fluorescence intensity time traces and time-resolved decay curves is demonstrated for synthetic as well as biological systems. By using continuous wave excitation and BIFL type detection, two dimensional fluorescence intensity time traces containing photons resolved in time with an accuracy of 50 ns and carrying polarization information can be recorded from individual immobilized molecules. In combination with specific analysis procedures, the SMS set-up is proved to be a suitable tool for the identification of different emitting species as well as for monitoring dynamical processes at the single molecule level.