Optimizing Spectral Differentiation in Super-Resolution Imaging via Chernoff Information

A fundamental goal in super-resolution microscopy is to simultaneously image multiple fluorescent species to visualize nanometer-scale protein interactions in real time. Previous work has demonstrated that spectral discrimination of diffraction-limited emitters can be achieved by placing a glass phase plate at the Fourier plane of the detection path in a wide-field fluorescence microscope, introducing a wavelength-dependent phase shift to the fluorescence emission to encode spectral information in an emitter’s point-spread function (PSF) [1]. However, as the emission spectra of different fluorophores become highly overlapping, the ability to accurately determine the fluorophore species decreases significantly. To address this, we adopted a Chernoff Information-based optimization scheme to determine the topography of several phase plate configurations to yield maximally distinguishable PSFs. We manufactured these phase plates and experimentally characterized our ability to determine an emitter’s three-dimensional location and fluorophore species, and explore the trade-off in precision between spatial localization and spectral classification.

[1] S. Fernando, et al., "Simultaneous spectral differentiation of multiple fluorophores in super-resolution imaging using a glass phase plate," Opt. Express  31, 33565-33581 (2023)