Review of the CH-STED performance and application range over conventional STED

Abstract

Super resolution in fluorescence through Stimulated Emission Depletion has been a reality since the beginning of the century. However, improvement is always pursued as the resolution is dependent on the intensity of the depletion beam. Combinations of depletion beam modulating masks are often proposed, yet their superposition is usually incoherent, and tends to lead to incidence of fluorescence in the zero-intensity area. Enhancements to the commonly used STED phase masks may provide sharper depletion areas without the need to significantly increase the depletion beam intensity. Coherent-Hybrid STED (CH-STED) is one alternative based on the addition of a 2D-STED phase mask to a rescaled z-STED mask to create a bi-vortex for improving contrast and axial resolution on the final image. This study evaluates the reproducibility and performance of CH-STED. The CH-STED mask was analyzed and modified to fit the existing STED microscope, despite certain setbacks. The manual and digital alignment of the depletion beam and the masks to ensure the zero-intensity area was performed with gold nanoparticles, taking advantage of their reflective property. Several samples, comprising of fluorescent beads and vimentin dyed in Mouse Embryonic Fibroblast cells, were subsequently imaged to provide optical sectioning and lateral resolution comparisons between 2D-STED and CH-STED. The removal of out-of-focus elements in thick sections of the cell samples was observed using the CH-STED mask, compared to 2D-STED where, regardless of depth, the out-of-focus elements still appeared. Further analysis was performed on the nanoparticle images to quantitatively establish the extent of the background suppression, as well as the improvement in the axial resolution in cell samples.