Fluorescence Lifetime Multiplex Imaging in Expansion Microscopy with Tunable Donor-Acceptor Polymer Dots

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in cell biology to detect biomolecules and their interactions. However, breaking the diffraction limit remains a challenge in FLIM due to the typically required photon counting method and the limited photon output of conventional dyes. Here, we introduce semiconducting polymer dots (Pdots) for fluorescence lifetime imaging in expansion microscopy by virtue of their tunable lifetime and huge photon budget. We developed three fluorescent Pdots with average lifetimes ranging from 0.4 to 5 ns by varying the polymer species and compositions. Despite their large spectral overlap, distinctive distributions of the Pdots can be resolved in the lifetime domain. The high fluorescence brightness and large photon output offered by Pdots enable multiplex lifetime imaging in photon-starved expansion microscopy, by which subcellular structures were resolved with a spatial resolution of ∼49 nm. This study reveals the potential of the tunable Pdot probes for lifetime multiplex imaging in expansion microscopy.