Highly multiplexed fluorescence microscopy with spectrally tunable semiconducting polymer dots

Current studies of biological tissues require visualizing diverse cell types and molecular interactions, creating a growing need for versatile techniques to simultaneously probe numerous targets. Traditional multiplexed imaging is limited to around five targets at once. Emerging methods using sequential rounds of staining, imaging, and signal removal can probe tens of targets but require specialized hardware and time-consuming workflows and face challenges with sample distortion and artifacts. We present a highly multiplexed fluorescence microscopy method using semiconducting polymer dots (Pdots) in a single round of staining and imaging. Pdots are small, bright, and photostable fluorescent probes with a wide range of tunable Stokes shifts (20 to 450 nanometers). Multiple series of Pdots with varying excitation wavelengths allow for fast (<1 minute) and single-round imaging of up to 21 targets in the brain and kidney. This method is based on a simple immunofluorescence workflow, efficient use of spectral space, standard hardware, and straightforward analysis, making it widely applicable for bioimaging laboratories.