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.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics ASCR v v i Vídeňská 1083 142 20 Prague 4 Czech Republic Microscopy Centre Institute of Molecular Genetics ASCR v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

Department of Biology of the Cell Nucleus Institute of Molecular Genetics ASCR v v i Vídeňská 1083 142 20 Prague 4 Czech Republic Microscopy Centre Institute of Molecular Genetics ASCR v v i Vídeňská 1083 142 20 Prague 4 Czech Republic Laboratory of Epigenetics of the Cell Nucleus Division BIOCEV Institute of Molecular Genetics of the ASCR v v i Průmyslová 595 252 50 Vestec Czech Republic

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