![]() ![]() Multiple techniques detect interactions between molecules on the nanometer scale, including fluorescent resonance energy transfer ( Zadran et al., 2012), fluorescence cross-correlation spectroscopy ( Fitzpatrick and Lillemeier, 2011), bimolecular protein complementation ( Kodama and Hu, 2012), and the proximity ligation assay ( Söderberg et al., 2006). Thus, researchers commonly investigate the localization of cellular components relative to each other using microscopy imaging to gain insights into biological function ( Lagache et al., 2015). As a general principle, the subcellular localization of biological components to specific domains is a crucial determinant of function, as spatial proximity allows for interactions among molecules, proteins, and organelles ( Yang, 2013). Microscopy imaging is a foundational technique for cell biology, as it allows for the interrogation of spatial relationships between cellular and tissue components and reveals previously unrecognized aspects of biological function ( Thorn, 2016). This article has an associated First Person interview with the first author of the paper. The SubcellularDistribution pipeline is designed to guide the user through the complete process of preparing image analysis data for publication, from image segmentation and data processing to visualization. Our open-source and freely available software detected RNA distributions comparably to commercially available image analysis software. Centrosomes are microtubule-organizing centers, and RNA enrichments at centrosomes are of emerging importance. To test the utility of the SubcellularDistribution pipeline, we examined the subcellular distribution of mRNA relative to centrosomes within syncytial Drosophila embryos. We generated an automated and customizable computational tool, the SubcellularDistribution pipeline, to facilitate object-based image analysis from three-dimensional (3D) fluorescence microcopy images. ![]() Understanding the underlying spatial relationships between objects through colocalization analysis of microscopy images is a fundamental approach used to inform biological mechanisms. The subcellular localization of objects, such as organelles, proteins, or other molecules, instructs cellular form and function. ![]()
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