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. 2021 Jun 2;8:674747. doi: 10.3389/fmolb.2021.674747

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Copyright © 2021 Hoyt.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Fundamentals of MOTiF imaging and spectral unmixing. With the MOTiF workflow, a tissue is stained with Opal fluorophores using a Leica BOND RX autostainer. The 6-plex, 7-color assay is then imaged using the Vectra Polaris slide scan protocol, wherein whole-slide scan images can be acquired in 10 min. Using inForm, designated library slides are used to isolate the exact spectral signature of each fluorophore to properly unmix each whole-slide composite image, as well as isolate and remove tissue autofluorescence. Spectral unmixing of signals is key to the Phenoptics technology and critical to ensuring accurate data for analysis.