New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-colour or hyperspectral SMLM can be particularly challenging due to non-linear image registration issues, which affect image quality and data interpretation. Many of these arise as a consequence of differences in illumination optics (beam profile, power density, polarization, point spread function) for the different light sources. This is particularly acute in evanescent-wave based approaches (TIRF) where beam shape, decay depth, and power density are important. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging.. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images with ~ 15-20 nm spectral resolution.. By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we identified an optimal fluorophore pair for two-colour SMLM. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20 and the peroxisomal protein, PMP70.
Hyperspectral super-resolution imaging with far-red emitting fluorophores using a thin-film tunable filter