Reduction of coherent artefacts in super‐resolution fluorescence localisation microscopy

Summary

Super‐resolution localisation microscopy techniques depend on uniform illumination across the field of view, otherwise the resolution is degraded, resulting in imaging artefacts such as fringes. Lasers are currently the light source of choice for switching fluorophores in PALM/STORM methods due to their high power and narrow bandwidth. However, the high coherence of these sources often creates interference phenomena in the microscopes, with associated fringes/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 localisation of the fluorophores, were characterised. Speckle phenomena degrade the resolution of the microscope at large length scales in reconstructed images, effects that were suppressed by the speckle scrambler, but the small length scale resolution is unchanged at ∼30 nm.

Lay description

Super‐resolution fluorescence localisation microscopy allows 20–30 nm resolution images to be created on fluorescently labelled specimens, which include intact biological cells. The diffraction limit of conventional optical microscopes is broken by building up the images point by point, localizing single fluorescent molecules in the image one by one. The majority of the fluorophores in the image are switched off (shelved in a dark state), whilst a small subset of them stochastically switch to a short lived fluorescent state so that their emission does not overlap. Such techniques have been very successful and led to the award of the Nobel Prize for chemistry in 2014. However, the lasers used to illuminate the specimen in such techniques can create artefacts in the images due to their high coherence. Light waves reflected from different parts of the microscope's optical components interfere with one another and create two closely related artefacts that reduce the quality of the images; fringes and speckle. Fringes are observed as stripes in the images (often with some curvature) and speckle patterns consist of a series of random blobs. Coherent light that is in phase causes the bright regions in the images (constructive interference), whereas out of phase light causes the dark regions (destructive interference). A large number of methods have been implemented in the literature with laser illuminated microscopes to remove such image artefacts. These include scanning the laser around the sample, launching the laser through a vibrated optical fibre and switching the laser on and off at high frequencies. We describe a new method to reduce coherent artefacts in optical microscopes, using a vibrating polymer membrane called a speckle scrambler. The speckle scrambler is demonstrated with super‐resolution fluorescence localisation microscopy. It performs well with this microscopy technique providing substantial improvements in resolution on intermediate length scales (μm), whereas the resolution at small length scales is unchanged (∼30 nm). The speckle scrambler is relatively easy to integrate in to the optical set up and compares favourably with alternative methods. Some of the measurements were performed with a total internal reflection fluorescence lens (TIRF). Such lenses are known to be particularly prone to coherence artefacts. The speckle scrambler also performed well with such TIRF lenses and thus appears also to be well suited to specialized single molecule experiments that use this type of apparatus.