Extracellular vesicles (EVs) function as important conveyers of information between cells and thus can be exploited as drug delivery systems or disease biomarkers. Transmission electron microscopy (TEM) remains the gold standard method for visualisation of EVs, however the analysis of individual EVs in TEM images is time-consuming if performed manually. Therefore, we present here a software tool for computer-assisted evaluation of EVs in TEM images. TEM ExosomeAnalyzer detects EVs based on their shape and edge contrast criteria and subsequently analyses their size and roundness. The software tool is compatible with common negative staining protocols and isolation methods used in the field of EV research; even with challenging TEM images (EVs both lighter and darker than the background, images containing artefacts or precipitated stain, etc.). If the fully-automatic analysis fails to produce correct results, users can promptly adjust the detected seeds of EVs as well as their boundaries manually. The performance of our tool was evaluated for three different modes with variable levels of human interaction, using two datasets with various heterogeneity. The semi-automatic mode analyses EVs with high success rate in the homogenous dataset (F1 score 0.9094, Jaccard coefficient 0.8218) as well as in the highly heterogeneous dataset containing EVs isolated from cell culture medium and patient samples (F1 score 0.7619, Jaccard coefficient 0.7553). Moreover, the extracted size distribution profiles of EVs isolated from malignant ascites of ovarian cancer patients overlap with those derived by cryo-EM and are comparable to NTA- and TRPS-derived data. In summary, TEM ExosomeAnalyzer is an easy-to-use software tool for evaluation of many types of vesicular microparticles and is available at http://cbia.fi.muni.cz/exosome-analyzer free of charge for non-commercial and research purposes. The web page contains also detailed description how to use the software tool including a video tutorial.

Centre for Biomedical Image Analysis Faculty of Informatics Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pharmacology and Immunotherapy Veterinary Research Institute vvi Brno Czech Republic

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$a Kotrbová, Anna $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

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$a TEM ExosomeAnalyzer: a computer-assisted software tool for quantitative evaluation of extracellular vesicles in transmission electron microscopy images / $c A. Kotrbová, K. Štěpka, M. Maška, JJ. Pálenik, L. Ilkovics, D. Klemová, M. Kravec, F. Hubatka, Z. Dave, A. Hampl, V. Bryja, P. Matula, V. Pospíchalová,

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$a Extracellular vesicles (EVs) function as important conveyers of information between cells and thus can be exploited as drug delivery systems or disease biomarkers. Transmission electron microscopy (TEM) remains the gold standard method for visualisation of EVs, however the analysis of individual EVs in TEM images is time-consuming if performed manually. Therefore, we present here a software tool for computer-assisted evaluation of EVs in TEM images. TEM ExosomeAnalyzer detects EVs based on their shape and edge contrast criteria and subsequently analyses their size and roundness. The software tool is compatible with common negative staining protocols and isolation methods used in the field of EV research; even with challenging TEM images (EVs both lighter and darker than the background, images containing artefacts or precipitated stain, etc.). If the fully-automatic analysis fails to produce correct results, users can promptly adjust the detected seeds of EVs as well as their boundaries manually. The performance of our tool was evaluated for three different modes with variable levels of human interaction, using two datasets with various heterogeneity. The semi-automatic mode analyses EVs with high success rate in the homogenous dataset (F1 score 0.9094, Jaccard coefficient 0.8218) as well as in the highly heterogeneous dataset containing EVs isolated from cell culture medium and patient samples (F1 score 0.7619, Jaccard coefficient 0.7553). Moreover, the extracted size distribution profiles of EVs isolated from malignant ascites of ovarian cancer patients overlap with those derived by cryo-EM and are comparable to NTA- and TRPS-derived data. In summary, TEM ExosomeAnalyzer is an easy-to-use software tool for evaluation of many types of vesicular microparticles and is available at http://cbia.fi.muni.cz/exosome-analyzer free of charge for non-commercial and research purposes. The web page contains also detailed description how to use the software tool including a video tutorial.

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$a Štěpka, Karel $u Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic.

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$a Maška, Martin $u Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic.

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$a Pálenik, Jakub Jozef $u Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic.

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$a Ilkovics, Ladislav $u Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

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$a Klemová, Dobromila $u Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

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$a Kravec, Marek $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

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$a Hubatka, František $u Department of Pharmacology and Immunotherapy, Veterinary Research Institute vvi, Brno, Czech Republic.

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$a Dave, Zankruti $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

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$a Hampl, Aleš $u Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

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$a Bryja, Vítězslav $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

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$a Matula, Pavel $u Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Brno, Czech Republic.

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$a Pospíchalová, Vendula $u Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.

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