Confocal Luminescence Lifetime Imaging with Variable Scan Velocity and Its Application to Oxygen Sensing

The dependence of the luminescence lifetime on the probe environment is the basis of a range of sensing techniques. The major advantage of using the lifetime as the sensitive parameter is its independence on the probe concentration. However, the instrumentation for lifetime measurements is complex, generally requiring time-resolved excitation and detection. Here, we present a simple method for the measurement of luminescence lifetimes on the microsecond scale based on variable excitation time determined by the scanning velocity. The technique is implemented in a confocal laser scanning microscope (CLSM), thus allowing not only simple lifetime measurement but also phosphorescence lifetime imaging. Since the method exploits the spatiotemporal dependence of sample excitation in a CLSM, there is no need for a pulsed or modulated light source or for additional time-resolved detection. The method can be realized in a standard CLSM without any modifications. The principle is demonstrated on oxygen sensing by collisional quenching of an oxygen-sensitive ruthenium(II) complex.