Print Email Facebook Twitter High precision wavefront control in point spread function engineering for single emitter localization Title High precision wavefront control in point spread function engineering for single emitter localization Author Siemons, M.E. (TU Delft ImPhys/Practicum support; Universiteit Utrecht) Hulleman, C.N. Thorsen, R.Ø. (TU Delft ImPhys/Quantitative Imaging) Smith, C.S. (University of Oxford) Stallinga, S. (TU Delft ImPhys/Imaging Physics) Department ImPhys/Imaging Physics Date 2018-04-02 Abstract 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. To reference this document use: http://resolver.tudelft.nl/uuid:7df262f9-ad12-4fbe-8b62-16b6c9189ab3 DOI https://doi.org/10.1364/OE.26.008397 ISSN 1094-4087 Source Optics Express, 26 (7), 8397-8416 Part of collection Institutional Repository Document type journal article Rights © 2018 M.E. Siemons, C.N. Hulleman, R.Ø. Thorsen, C.S. Smith, S. Stallinga Files PDF oe_26_7_8397.pdf 4.32 MB Close viewer /islandora/object/uuid:7df262f9-ad12-4fbe-8b62-16b6c9189ab3/datastream/OBJ/view