Optically Switchable CLEAR Probes Enable Rapid, Biocompatible and High-Efficiency Fluorophore Exchange for Ultra-Plex, High-Resolution Immunofluorescence Imaging

Abstract

Immunofluorescence imaging remains a foundational technique in biological research, enabling visualization of protein targets within native environments. However, the ability of conventional immunofluorescence approaches to fully capture biological complexity is limited by low multiplexing capacity, typically restricted to four or five targets due to spectral overlap among fluorophores. Recent advances, including DNA-labelled antibodies or chemical fluorescence ablation, aim to enhance multiplexing but face considerable challenges. DNA-based approaches require non-conventional reagents and are operationally complex, while chemical clearing methods are time-intensive and risk sample damage from harsh reagents. To address these limitations, we introduce CLEAR (Cleavable Light-Erased Antibody Reporter) probes—a simple, rapid, and broadly applicable platform for scalable multiplexing using conventionally conjugated antibodies. Through a highly optimized bottom-up molecular design, CLEAR probes deliver one of the fastest and most efficient signal-clearing strategies, achieving >98% fluorescence removal in under two minutes using a mild, non-toxic light dose. This allows their integration into iterative immunostaining workflows and, importantly, preserves sample and image quality across multiple staining cycles for supporting high-throughput, potentially unlimited multiplexing. Moreover, the excellent biocompatibility of this approach enables its extension from fixed to live-cell and tissue imaging. Its compatibility with diverse fluorophores makes it readily adaptable to advanced imaging modalities, including super-resolution microscopy. Finally, we demonstrate the synergistic potential of the CLEAR platform—where ultrafast, efficient, and biocompatible fluorophore clearance allows high-dimensional interrogation of coordinated cytoskeletal remodeling and organelle redistribution during dynamically regulated immunological synapse formation.