title: Correcting chromatic offset in multicolor super-resolution localization microscopy.
abstract: Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK. meerdelyi@gmail.com
authors.forename: Miklos
authors.initials: M
authors.lastname: Erdelyi
authors.forename: Eric
authors.initials: E
authors.lastname: Rees
authors.forename: Daniel
authors.initials: D
authors.lastname: Metcalf
authors.forename: Gabriele S Kaminski
authors.initials: GS
authors.lastname: Schierle
authors.forename: Laszlo
authors.initials: L
authors.lastname: Dudas
authors.forename: Jozsef
authors.initials: J
authors.lastname: Sinko
authors.forename: Alex E
authors.initials: AE
authors.lastname: Knight
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
mesh_terms: D000465:Algorithms; D016477:Artifacts; D003124:Colorimetry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*