Accurate gaze direction measurements with free head movement for strabismus angle estimation.

We present the Delft Assessment Instrument for Strabismus in Young children (DAISY) a device designed to measure angles of strabismus in young children fast and accurately. DAISY allows for unrestrained head movements by the mean of a triple camera vision system that simultaneously estimates the head rotation and the eye pose. The device combines two different methods to record bilateral eye position: corneal reflections (Purkinje images) and pupillary images. Detailed results are provided on three orthotropic subjects (age 25-27). Three different conditions were tested: 1) gaze ahead, 2) gaze ahead with different head rotations, and 3) fixed head with different eye positions. Systematic errors occurred between subjects that need further study. The system reached sufficient accuracy to be applied for the measurement of angles of strabismus, almost independent from the head pose.

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines.