AutoFoci, an automated high-throughput foci detection approach for analyzing low-dose DNA double-strand break repair.

Double-strand breaks (DSBs) are the most lethal DNA damages induced by ionising radiation (IR) and their efficient repair is crucial to limit genomic instability. The cellular DSB response after low IR doses is of particular interest but its examination requires the analysis of high cell numbers. Here, we present an automated DSB quantification method based on the analysis of γH2AX and 53BP1 foci as markers for DSBs. We establish a combination of object properties, combined in the object evaluation parameter (OEP), which correlates with manual object classification. Strikingly, OEP histograms show a bi-modal distribution with two maxima and a minimum in between, which correlates with the manually determined transition between background signals and foci. We used algorithms to detect the minimum, thus separating foci from background signals and automatically assessing DSB levels. To demonstrate the validity of this method, we analyzed over 600.000 cells to verify results of previous studies showing that DSBs induced by low doses are less efficiently repaired compared with DSBs induced by higher doses. Thus, the automated foci counting method, called AutoFoci, provides a valuable tool for high-throughput image analysis of thousands of cells which will prove useful for many biological screening approaches.

A Model based Survey of Colour Deconvolution in Diagnostic Brightfield Microscopy: Error Estimation and Spectral Consideration.

Colour deconvolution is a method used in diagnostic brightfield microscopy to transform colour images of multiple stained biological samples into images representing the stain concentrations. It is applied by decomposing the absorbance values of stain mixtures into absorbance values of single stains. The method assumes a linear relation between stain concentration and absorbance, which is only valid under monochromatic conditions. Diagnostic applications, in turn, are often performed under polychromatic conditions, for which an accurate deconvolution result cannot be achieved. To show this, we establish a mathematical model to calculate non-monochromatic absorbance values based on imaging equipment typically used in histology and use this simulated data as the ground truth to evaluate the accuracy of colour deconvolution. We show the non-linear characteristics of the absorbance formation and demonstrate how it leads to significant deconvolution errors. In particular, our calculations reveal that polychromatic illumination causes 10-times higher deconvolution errors than sequential monochromatic LED illumination. In conclusion, our model can be used for a quantitative assessment of system components--and also to assess and compare colour deconvolution methods.

Analysis of fluid-phase endocytosis in (intact) plant cells.

Endocytosis is a continuous process at the plasma membrane at least of all eukaryotic cells. Regardless of the molecular machinery, which drives the formation and uptake of endocytic vesicles, it is reasonable to assume that this process inevitably collects external fluid. Hence, at least for the majority of apoplastic solutes, the endocytosis of the fluid phase is likely to be an inevitable process. Due to its independence from the molecular machinery and low selectivity with respect to the cargo, it is thus perfectly suited to be used as a tracer to follow the activity of all endocytic events. Here we describe simple protocols based on fluorescence microscopy, which yield quantitative information about endocytic vesicle sizes-with sub-diffraction accuracy-as well as the size exclusion limits for these uptake routes.