Wash-Free Live-Cell Imaging Using Homogeneous Immunosensors

Imaging membrane proteins on the cell surface is crucial for studying cellular functions involving their mobility, localization, and clustering. Conventional antibody-based fluorescence imaging requires multiple washing steps to reduce background signals from unbound fluorescent antibodies. These steps stress living cells and potentially alter their characteristics and behavior. To address this, we propose a protocol for wash-free fluorescence imaging using an immunosensor, the Quenchbody (Q-body), which emits fluorescence upon antigen binding, eliminating the need for washing steps. In this chapter, we introduce the preparation of a Q-body against a cancer biomarker, human epidermal growth factor receptor 2 (HER2), and its application in live-cell imaging by simply adding to the culture medium without washing. This tool offers a minimally invasive and efficient approach to real-time membrane protein analysis. Importantly, because the antibody component is exchangeable with other target-specific antibodies, Q-bodies are adaptable for detecting a wide range of membrane proteins in live-cell imaging.