Correction of bleaching artifacts in high content fluorescence correlation spectroscopy (HCS-FCS) data

ABSTRACT We developed an automated approach to Fluorescence Cross -Correlation Spectroscopy (FCCS) to screen for molecular interactions in living cells (HCS -FCS). Similar to manual measurements , we had to address the effects of measurement artifacts. In this paper we use d wavelet shrinking to correct for bleaching artifacts. Wit hout correction, bleaching of both dyes used for cross-correlation measurements result ed in a cross-correlation signal indicating mol ecular interaction even if there was no interaction present in the sample. We used simulations to develop our new method. To evaluate our new approach in vivo we used two control strains in S. cerevisiae . The negative control contained the non -interacting fluorescence proteins eGFP and mCherry. As the positive control we used a tandem of these two proteins. We measured more than 2500 individual cells and demonstrated that we could reduce the erroneous cross-correlation signal of the negative control from 69% to 4% compared to the cross-correlation signal of the positive control in the mCherry channel and from 24% to 5% for the eGFP channel . Keywords: Fluorescence Correlation Spectros copy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), Wavelet, Bleaching , High-Content Screening , Machine Learning .