Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

Abstract

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutaminemediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington's Disease (HD) model. © Springer Science+Business Media, LLC 2010Correspondence to: Pengyu Hong, hongpeng@brandeis.edu. NIH Public Access Author ManuscriptNeuroinformatics. Author manuscript; available in PMC 2011 January 18. has been adopted to conduct analysis of primary neurons which have highly complex cellular morphologies and branching patterns. This type of analysis has been a powerful methodology in RNA interference (RNAi) screening experiments to identify genes that are important for neuronal development, physiology and disease (Dragunow 2008;Sepp et al. 2008). Such studies are expected to be an increasingly utilized approach in drug discovery as neuronal morphology can be used as an assay for cell health.Noise and complex signals make it challenging to quantify neurites and neuromeres, which are two essential morphological elements that relate to neuronal health status. For example, noise can arise from non-neuronal cells, cell debris, or limitations in the sensitivity of charge-coupled device (CCD) cameras that are commonly used to collect images. The signal levels of key neurites can be very weak because of various factors, including variations in the thickness of the neurites, sub-optimal focus plane established by automated microscopy, 3-dimensional nature of the cell cultures, and inadequate exposure time lengths. In general, microscope settings for a screen are established on the basis of what is optimal for an average well of a multiwell screening plate. However, cell profiles in some wells may be significantly different in marker intensities, which can lead to under-or over-e...

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