Fluorescence Lifetime Imaging Microscopy (FLIM) on the Single Molecule Level

摘要

During the last years, several methods for the detection of individual fluorescently labeled molecules have been developed. Especially, for the study of inhomogeneous complex systems single molecule spectroscopy can provide information that is difficult to obtain from ensemble measurements. Due to the limited observation time of individual freely diffusing molecules in solution fluorescence scanning systems including single molecule search programs have been developed to monitor the dynamical behavior of individual immobilized probe molecules on surfaces (for review). Besides polarization measurements, so far only the spectral characteristics, i.e. different emission maxima of different dyes, have been used to detect, identify, and monitor the dynamics of individual fluorescent molecules on surfaces. Therefore we decided to further extend the number of obtainable parameters by developing a scanning system for time-correlated single-photon counting (TCSPC). As shown in Fig. 1 the system consists essentially of an inverted confocal fluorescence microscope and a motion driven controller x,y-microscope stage (SCAN 100x100 and MC2000; Maerzhaeuser, Germany). As excitation source we used a pulsed diode laser emitting at 640 nm with a repetition rate of 50 MHz. The laser diode was driven by an external pulse generator.