Microarray analysis at single-molecule resolution.
Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state-of-the-art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack of appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of 2-20 microm and quantifies the abundance of target molecules by determining average pixel intensities, a novel high-resolution approach directly visualizes individual bound molecules as diffraction-limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. First, it consists of a single-molecule detection step, based on undecimated wavelet transforms, and second, a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micrometer and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15, the false negatives relative error was below 15%. Separation of foreground/background is proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements.
Mureşan
Leila
L
Department of Knowledge-Based Mathematical Systems, Johannes Kepler University, Linz 4040, Austria. leila.muresan@jku.at
Jacak
Jarosław
J
Klement
Erich Peter
EP
Hesse
Jan
J
Schütz
Gerhard J
GJ
D000465:Algorithms; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D015233:Models, Statistical; D010316:Particle Size