BACKGROUND: High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology. RESULTS: We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach. CONCLUSIONS: Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.

We investigate different approaches for efficient feature space reduction and compare different methods for cell classification. The application context is the development of automatic methods for analysing fluorescence microscopy images with the goal to identify those genes that are involved in the mitosis of human cells (cell division). We distinguish four cell classes comprising interphase cells, mitotic cells, apoptotic cells, and cells with clustered nuclei. Feature space reduction was performed using the Principal Component Analysis and Independent Component Analysis methods. Six classification methods were examined including unsupervised clustering algorithms such as K-means, Hard Competitive Learning, and Neural Gas as well as Hierarchical Clustering, Support Vector Machines, and Random Forests classifiers. Detailed results on the cell image classification accuracy and computational efficiency achieved using different feature sets and different classification methods are reported.