Through‐Bond Energy Transfer Cassette with Dual‐Stokes Shifts for “Double Checked” Cell Imaging

Organic dyes generally suffer from small Stokes shift that usually leads to self-quenching and -gaining errors during the fluorescent imaging process. Here, a through-bond energy transfer (TBET) cassette is developed with large Stokes shift to pursue precise cell imaging. The TBET is constructed by covalently conjugated tetraphenylethene (acts as donor) and rhodamine (acceptor) through an acetylene bond. The constructed TBET cassette distinctly behaves as dual-Stokes shifts, including a large pseudo-Stokes shift caused by energy transfer, from donor's absorption to acceptor's emission (up to 260 nm) and a smaller Stokes shift of acceptor molecules itself. Due to the intrinsic dual-Stokes shifts, TBET cassette exhibits specific "dual distinct absorbances, single shared emission" properties, which can be excitated under two different laser channels. By colocalization of the imaging readouts of these two channels, the precisely "double checked" fluorescent imaging is achieved in living cells.