Traditional fluorescence microscopy is typically limited to 4–5 imaging channels due to physical constraints from excitation sources, broad dye spectra, and available optical filters, making further multiplexing both technically challenging and costly. Increasing the number of resolvable channels would yield exponential gains in biological information and reduce time spent on sample preparation, imaging, and downstream processing. Our goal is to provide an effective, high-throughput, multiplexed fluorescence approach with minimal system constraints, while demonstrating a powerful molecular tool for studying complex biological systems. We present a rapid line-scan spectral microscopy platform that leverages polymer dot probes to achieve highly multiplexed fluorescence imaging. These polymer dots are bright, photostable, and exhibit narrow emission spectra with tunable Stokes shifts determined by polymer lengths, making them ideal for multiplexed imaging. Using these probes and a single 405 nm excitation source, our system currently achieves 8-channel imaging in multiplexed, antibody-labeled 50 μm mouse kidney sections, with plans to expand to 11 distinct channels at 405 nm and extend to 488 nm and 561 nm excitable polymer dot series. To enhance performance, we incorporated iterative spectral unmixing algorithms and GPU-accelerated processing for improved accuracy and significantly reduced processing time, and custom Python-controlled hardware triggering for 40 Hz line scans. This approach substantially expands the multiplexing capacity of fluorescence microscopy and enables high-throughput imaging of biological systems for diverse biomedical research applications.
Chris Kim (Sun,) studied this question.