Summary Single-molecule spectroscopy and detection are powerful techniques for the study of single fluorescent particles and their interaction with their environment. We present a low-cost system for simultaneous real-time acquisition, storage of inter-photon arrival times and the calculation and display of the fluorescence time trace, autocorrelation function and distribution of delays histogram for single-molecule experiments. From a hardware perspective, in addition to a multi-core computer, only a standard low-cost counting board is required as processing is software-based. Software is written in a parallel programming environment with time crucial operations coded in ANSI-C. Crucial to system performance is a simple and efficient real-time autocorrelation algorithm (acf) optimized for the count rates (approximately 104 cps) encountered in single-molecule experiments. The algorithm's time complexity is independent of temporal resolution, which is maintained at all time delays. The system and algorithm's performance was validated by duplicating the signal from the photon detector and sending it to both the ordinary counter board and a commercial correlator simultaneously. The data acquisition system's robustness under typical single-molecule experimental conditions was tested by observing the diffusion of Rhodamine 6G molecules in deionized water.
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