Abstract
In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders’ colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders’ colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.
Highlights
In biomedical studies, the colocalization is commonly understood as the overlap between signals produced by distinctive dyes or stains in images
To demonstrate the performance of the proposed coefficients for electron microscopy (EM), we investigated the level of the spatial interaction of phosphatidylinositol 4,5-bisphosphate (PIP2) with fibrillarin in nucleoli and conceptually compared the results with the Manders’ colocalization coefficients and distributional data for fluorescent imaging
Fibrillarin localizes at the boundary between fibrillar centers (FCs) and the dense fibrillar component (DFC), which is a site of ribosomal DNA transcription, and in the DFC itself, where pre-rRNA processing occurs (Boisvert et al 2007; Hernandez-Verdun 2006, 2011; Hozák et al 1994; Ochs et al 1985; Sobol et al 2013)
Summary
The colocalization is commonly understood as the overlap between signals produced by distinctive dyes or stains in images This term is currently associated especially with evaluating the immunostaining in images acquired using fluorescence microscopy (FM). Histochem Cell Biol (2016) 146:391–406 of thresholding, parameter settings, and colocalization method, Dunn et al 2011) Another issue represents the subjective or algorithmic selection of the region of interest (Jaskolski et al 2005; Lachmanovich et al 2003; Ramirez et al 2010). The merged fluorescent images can result in optical illusions, which in turn represent the misleading puzzle for the human visual perception and brain All these factors may negatively bias the assessment of the colocalization and influence scientific conclusions
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