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Technical Report

TEM ExosomeAnalyzer: a computer-assisted software tool for quantitative evaluation of extracellular vesicles in transmission electron microscopy images

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Article: 1560808 | Received 31 May 2018, Accepted 03 Dec 2018, Published online: 21 Jan 2019
 

ABSTRACT

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.

Acknowledgments

We thank Oncogynaecologic Center, University Hospital Brno, CZ, especially to medical doctors Vít Weinberger, Igor Crha, Luboš Minář, Markéta Bednaříková and Eva Jandáková, for patient ascites and plasma samples.

We also thank to Central European Institute of Technology Core Facility of Cryo-Electron Microscopy and Tomography supported by the Czech Infrastructure for Integrative Structural Biology research project (LM2015043 funded by the Ministry of Education, Youth and sports of the Czech Republic) for their assistance in obtaining the scientific data presented in this paper. Especially, we thank to Anatolij Filimonenko for technical assistance with osmium tetroxide staining.

Disclosure statement

The authors reported no potential conflict of interest.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Masaryk University [grant number MUNI/M/1050/2013], [grant number MUNI/E/0876/2018]; the Czech Science Foundation [grant number 17-11776Y]; and the Ministry of Education, Youth and Sports of the Czech Republic (Czech-BioImaging projects) [grant number LM2015062], [grant number CZ.02.1.01/0.0/0.0/16_013/0001775]. FH was supported by the Ministry of Education, Youth and Sports OPVVV PO1 project “FIT” (Pharmacology, Immunotherapy, nanoToxicology) [grant number CZ.02.1.01/0.0/0.0/15_003/0000495] .

Notes on contributors

Anna Kotrbová

VP, VB and PM conceived and designed the research. AK, ZD and VP isolated the EVs, AK and VP served as experts for EVs annotation and evaluated the performance of TEM ExosomeAnalyzer. DK, LI and AH performed the TEM experiments using a Morgagni 268D electron microscope. MK performed all analyses using a Tecnai G2 transmission electron microscope, including cryo-EM. KŠ, MM and JJP developed and tested the software tool as well as designed and made the web page. KŠ, AK, ZD and VP made the video tutorial. FH performed NTA and TRPS measurements and prepared liposomes. AK, MM, KŠ, PM and VP analysed the data. AK, KŠ, VP, VB and PM interpreted the data and wrote the manuscript. Each author reviewed and made critical comments to the manuscript.