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Synthetic and genetically encoded optical switch probes and lock-in detection methods provide a means to modulate and isolate fluorescence signals from the optical switch and protein complexes of interest against un-modulated background sources. In this paper we propose an improved method for optical lock-in detection (OLID) fluorescence microscopy that can significantly enhance image contrast of optical switch probes within living cells. In particular, we introduce a method for automatic and optimal determination of the reference waveform, which is consequently used for the correlation analysis and construction of the contrast-enhanced image. The method involves computing the average scope of each pixel in the image over several switching cycles and then selecting the pixel with maximum scope as a reference point. The correlation between the fluorescence emission of each pixel over the switching cycles and the reference waveform is then computed. Finally a new contrast-enhanced image is constructed based on the scope-weighted correlation value. Our results of analysis show that this method and algorithm can remarkably improve contrast enhancement and noise reduction compared to the original method.
Date of Conference: 18-20 June 2010