Nanofibers are currently among the most researched nanomaterials in materials science. Various high-resolution microscopy techniques are used for morphological investigations, with the diameter as primary characteristic. Since methodological factors influencing the diameter distribution are usually ignored, numerical values can hardly be compared across different or even within single studies. Here, we investigate influencing factors such as microscopy technique, degree of magnification, eventual coatings, and the analysts' bias in the image selection and evaluation. We imaged a single nanofiber sample using scanning electron microscopy (SEM), helium ion microscopy (HIM), atomic force microscopy (AFM), and transmission electron microscopy (TEM). These techniques yield significant methodological variations between the diameter distributions. We further observed a strong influence of analysts' subjectivity, with a consistent average deviation between 4 different analysts of up to 31%. The average deviation between micrographs within each category was 14%, revealing a considerable influence of micrograph selection and strong potential for cherry picking. The mean values were mostly comparable with the results using automated image analysis software, which was more reproducible, much faster, and more accurate for images with lower magnification. The results demonstrate that one of the most frequently measured characteristics of nanofibers is subject to strong systematic fluctuations that are rarely if ever addressed.
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