As described above, great care in the experimental setup is given to the high precision and repeatability of the beam positioning and focusing across the entire range of delay stage settings. In order to achieve this goal, a setup including several pinholes for spatial filtering had been chosen. This practice, however, leads to a variation of the stimulation beam intensity at sample position of approximately 25% between the shortest and the longest time delay (3.3 ns). In order to compensate for this variation in intensity, two images have been recorded at each delay stage position, one with the excitation beam enabled and the other without. The measured signal strength, at a given temporal delay , gives Display Formula
(2)where is proportional to the number of excited molecules and Display Formula
(3)for a disabled excitation beam. The signal of interest Display Formula
(4)can hence be estimated as Display Formula
(5)All LDFLI-measured fluorescence decay curves shown below were obtained using the full LDFLI setup including the FV300 scan unit. For R6G no image scans were performed. Instead, the signals were recorded at a fixed position on the sample. The ICG image intensity curves in Fig. 2 were obtained by recording LDFLI images of two different two-dimensional ICG samples using the FV300 scan unit and the Olympus Fluoview software and then determining the average pixel intensity inside the regions of interest marked in Fig. 3 and Fig. 4 of the individual images with the help of ImageJ. The procedure was repeated for different regions of interest, which yielded comparable results. The time delay control between the excitation and stimulation beams for all measurements was realized by mechanically moving a retro-reflector in the delay line of the stimulation beam. To enable the use of a lock-in amplifier for signal discrimination, the excitation beam was modulated at a frequency of 720 Hz using a mechanical chopper. Initial experiments using an electro-optical modulator yielded worse signal-to-noise ratios, even at significantly higher modulation frequencies up to 25 KHz. The degradation of ratio may be attributed to the reduced modulation depth of 10 to 50% relative to the chopper’s 100% modulation depth. In addition, the relatively low modulation rate of the chopper compared to the high laser repetition rates led to sufficient data point averaging in order to diminish pulse-to-pulse laser power fluctuation effects. The lock-in amplifier signal was either directly recorded or, for the image recording, fed into the scan unit’s control computer.