A novel algorithm, Adjacent Pixel Temporal Intensity Correlation (APTIC), was developed to detect single fluorescent molecules by their stochastic emission patterns; photoblinking and photobleaching. The algorithm was evaluated using simulated image data and Total Internal Reflection Fluorescence Microscopy (TIRF-M) to count the number fluorescently labelled protein molecules adsorbed onto glass substrates modified by Radio Frequency Glow Discharge (RFGD) deposition. By selecting an appropriate correlation threshold, the algorithm was capable of detecting synthetic flashes with a signal-to-noise ratio (SNR) as low as 2.0 with 90% sensitivity. The methodology holds great promise for mapping the amount and distribution of biomolecules on surfaces.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.