Analyzing spectral heterogeneity of individual fluorophores using DWP-enhanced single-molecule spectroscopy

We introduce a four dual wedge prisms (DWPs) design for high-throughput single-molecule spectroscopy in single-molecule localization microscopy (SMLM). This system enhances photon collection and spectral resolution, enabling detailed analysis of dye spectral heterogeneity. Our findings reveal significant spectral variability among fluorophores, critical for molecular diagnostics, biological imaging, and materials science. By characterizing this heterogeneity, we can select stable fluorophores with minimal overlap and explore environmental factors affecting spectral properties, ensuring reliable results. Leveraging this variability also enhances multiplexed imaging and novel sensing mechanisms. This presentation will cover the optical setup, data acquisition, and implications for single-molecule spectroscopy.