biostudies-literatureBarbotin AEPA Cephalosporin FundH2020 Marie Sklodowska-Curie ActionsDeutsche ForschungsgemeinschaftEuropean Research CouncilWolfson FoundationJohn Fell Fund, University of OxfordMedical Research CouncilJena Center of Soft MatterWellcome TrustBiotechnology and Biological Sciences Research CouncilEngineering and Physical Sciences Research Council1742-1753https://www.ebi.ac.uk/biostudies/studies/S-EPMC7366504biostudies-literatureUnknown7(7)Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale.ACS photonicsBackground Reduction in STED-FCS Using a Bivortex Phase Mask.PMC7366504707348MC_UU_12010/9EP/L016052/1104924/14/Z/14MR/K01577X/1091911MC_UU_00008/9203285BB/L00433X/1G0902418203285/C/16/Z695140MC_UU_12010BB/P026354/1MC_UU_12025Urbancic IEggeling CBooth MBarbotin AGaliani SfalseBackground Reduction in STED-FCS Using a Bivortex Phase Mask.Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale.2020-01-01T00:00:00Z2020 Jul2025-04-05T13:48:28.287Z2025-04-05T13:48:28.287ZS-EPMC73665043268560910.1021/acsphotonics.0c00388