Speckle scrambler for improved super-resolution fluorescence localisation microscopy

Super-resolution localization microscopy techniques depend on uniform illumination across the field of view, otherwise the resolution is degraded, resulting in imaging artefacts such as stripes. Lasers are currently the light source of choice for switching fluorophores in PALM/STORM methods due to their high powers and narrow bandwidths. However, the high coherence of these sources often creates interference phenomena in the microscopes, with associated fringe/speckle artefacts in the images. We quantitatively demonstrate the use of a polymer membrane speckle scrambler to reduce the effect of the coherence phenomena. The effects of speckle in the illumination plane, at the camera and after software localization of the fluorophores were characterized. Speckle phenomena degrade the resolution of the microscope at large length scales in reconstructed images, effects that are suppressed by the speckle scrambler, but the small length scale resolution is unchanged at ~30 nm.