Ultra-fast cycling for multiplexed cellular fluorescence imaging

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Here we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz) / trans-cyclooctene (TCO) mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t_1/2 < 1 sec) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.