Point spread function (PSF) engineering is used in single emitter localization to measure the emitter position in 3D and possibly other parameters such as the emission color or dipole orientation as well. Advanced PSF models such as spline fits to experimental PSFs or the vectorial PSF model can be used in the corresponding localization algorithms in order to model the intricate spot shape and deformations correctly. The complexity of the optical architecture and fit model makes PSF engineering approaches particularly sensitive to optical aberrations. Here, we present a calibration and alignment protocol for fluorescence microscopes equipped with a spatial light modulator (SLM) with the goal of establishing a wavefront error well below the diffraction limit for optimum application of complex engineered PSFs.We achieve high-precision wavefront control, to a level below 20 mλ wavefront aberration over a 30 minute time window after the calibration procedure, using a separate light path for calibrating the pixel-to-pixel variations of the SLM, and alignment of the SLM with respect to the optical axis and Fourier plane within 3 μm (x/y) and 100 μm (z) error. Aberrations are retrieved from a fit of the vectorial PSF model to a bead z-stack and compensated with a residual wavefront error comparable to the error of the SLM calibration step. This well-calibrated and corrected setup makes it possible to create complex '3D+λ' PSFs that fit very well to the vectorial PSF model. Proof-of-principle bead experiments show precisions below 10 nm in x, y, and λ, and below 20 nm in z over an axial range of 1 μm with 2000 signal photons and 12 background photons.

We show that the position of single molecules in all three spatial dimensions can be estimated alongside its emission color by diffractive optics based design of the Point Spread Function (PSF). The phase in a plane conjugate to the aperture stop of the objective lens is modified by a diffractive structure that splits the spot on the camera into closely spaced diffraction orders. The distance between and the size of these sub-spots are a measure of the emission color. Estimation of the axial position is enabled by imprinting aberrations such as astigmatism and defocus onto the orders. The overall spot shape is fitted with a fully vectorial PSF model. Proof-of-principle experiments on quantum dots indicate that a spectral precision of 10 to 20 nm, an axial localization precision of 25 to 50 nm, and a lateral localization precision of 10 to 30 nm can be achieved over a 1 μm range of axial positions for on average 800 signal photons and 17 background photons/pixel. The method appears to be rather sensitive to PSF model errors such as aberrations, giving in particular rise to biases in the fitted wavelength of up to 15 nm.