Multicolor 4Pi single-molecule localization based on differences in interference patterns
Published in Optics Letters , 2025
4Pi single-molecule localization microscopy (4Pi-SMLM) achieves sub-10 nm isotropic three-dimensional resolution, representing a signiffcant advancement in super-resolution imaging. However, traditional multicolor imaging techniques generally require additional modiffcations to the optical path, which complicates the system and leads to photon loss. In this study, we introduce a new, to the best of our knowledge, multicolor imaging method that leverages the differences in 4Pi-PSF size and interference fringe spacing caused by wavelength variations, enabling multicolor 4Pi-SMLM imaging without hardware modiffcations. This method captures color information directly from the raw data by utilizing wavelength-dependent point spread functions (PSFs). By globally fftting the data within multiple interference channels, we could achieve both high localization accuracy and color separation accuracy for single molecules of different colors.