Simultaneous spectral differentiation of multiple fluorophores in super-resolution imaging using a glass phase plate

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super -resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved-21-nm lateral localization precision (FWHM) and-17-nm axial precision (FWHM) with an average of 1,800 -3,500 photons per PSF and a background as high as 130 -400 photons per pixel. The modified PSF distinguished fluorescent probes with-80 nm separation between spectral peaks.(c) 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

Automated summary

By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. In this study, we inserted a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to generate distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrated that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be used for hyper-spectral super-resolution microscopy of biological samples.