Engineering Point Spread Functions using a Single Glass Phase Plate for 4D Super-resolution Fluorescence Microscopy

Multicolor localization microscopy typically relies on sequential imaging and bandpass filters to distinguish

fluorescent tags, which introduce temporal delays specially during live cell imaging, and wastes precious

photons. Alternatively, multiple fluorophore species can be simultaneously imaged and distinguished using

engineered point-spread functions (PSF) that imprint unique patterns sensitive to the spatial coordinates and

the emission wavelength of a fluorophore. Here, we insert a silicon-dioxide phase plate with only four

thickness regions at the Fourier plane of the detection path of a wide-field fluorescence microscope to

produce distinguishable PSFs (X-PSFs) at different wavelengths with sufficient 3D spatial localizability. We

localize the X-PSFs both spatially and spectrally using a Maximum Likelihood Estimation algorithm with a

faster Gaussian Cubature approximation than the standard Fourier Transforms to calculate the diffraction

integral. We demonstrate two-color and three-color super-resolution dSTORM imaging of fixed U2OS cells,

in which the peak-to-peak separation between consecutive spectra was ∼80 nm. All fluorophore species were

excited simultaneously and imaged without emission filters. The X-PSF achieves ∼21 nm lateral localization

precision, ∼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 can distinguish up to three fluorescent

probes with ∼80 nm peak-to-peak separation between consecutive spectra.