Single-virus tracking (SVT) offers the opportunity to monitor the journey of individual viruses in real time and to explore the interactions between viral and cellular structures in live cells, which can assist in characterizing the complex infection process and revealing the associated dynamic mechanisms. However, the low brightness and poor photostability of conventional fluorescent tags (e.g., organic dyes and fluorescent proteins) greatly limit the development of the SVT technique, and challenges remain in performing multicolor SVT over long periods of time. Owing to the outstanding photostability, high brightness and narrow emission with tunable color range of quantum dots (QDs), QD-based SVT (QSVT) enables us to follow the fate of individual viruses interacting with different cellular structures at the single-virus level for milliseconds to hours, providing more accurate and detailed information regarding viral infection in live cells. So far, the QSVT technique has yielded spectacular achievements in uncovering the mechanisms associated with virus entry, trafficking and egress. Here, we provide a detailed protocol for QSVT implementation using the viruses that we have previously studied systematically as an example. The specific procedures for performing QSVT experiments in live cells are described, including virus preparation, the QD labeling strategies, imaging approaches, image processing and data analysis. The protocol takes 1-2 weeks from the preparation of viruses and cellular specimens to image acquisition, and 1 d for image processing and data analysis.
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