Precisely Localizing Wavelength Sensitive Point-Spread Functions Engineered With a Silicon Oxide Phase Plate

Recently point-spread function (PSF) engineering has made important contributions to single molecule localization microscopy (SMLM). PSF engineering has allowed scientists to encode information into the PSF in addition to position. One drawback of the current work is that it typically uses expensive polarization dependent spatial light modulators to modify the phase of the signal. Here, we introduce a simple silicon oxide phase plate that, when placed in the Fourier plane of a microscope, makes the PSF very sensitive to wavelength. We have used this engineered PSF (ePSF) to identify different species of fluorescent nano-spheres based only on the form of their respective PSFs. This experiment was done on a home-built microscope. Both the spatial and wavelength localization precisions measured from this experiment were in the single nanometer range for ~5000 photons. We have also used this same phase plate to do two-color SMLM. This imaging was done on fixed B2SO cells. We were able to resolve microtubules to 50 nm. This experiment was done on a commercially available microscope, the Vutara 352. In the future we aim to use our phase plate to look at small spectral shifts in SMLM dyes caused by environmental conditions, such as pH, within the cell.