A Glass Phase Plate for Wavelength Sensitive Superresolution Microscopy

Abstract

Precision in superresolution microscopy is dependent on the photon yield. Multicolor localization microscopy typically relies on bandpass filters and sequential imaging to distinguish fluorescent tags. By engineering the pointspread function with a spatial light modulator (SLM), the wavelength of specific fluors can be distinguished by their unique pattern on the camera face and can thereby increase photon budgets and be imaged simultaneously. Here, instead of an SLM, we insert a silicon-dioxide phase plate, the X-phase plate (XPP) in the Fourier plane of the detection path of a wide-field fluorescence microscope to engineer the PSFs (XPSF). We demonstrate that the XPSF can be used for hyper-spectral superresolution microscopy of biological samples. The XPSF achieves ∼25 nm in-plane resolution, ∼250 nm axial resolution and can distinguish three fluorescent probes with ∼80 nm peak separation between consecutive spectra at the vicinity of axial focus.