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.