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.

中文翻译：

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使用 Bivortex 相位掩模减少 STED-FCS 中的背景。

荧光相关光谱 (FCS) 是研究活细胞分子动力学的重要工具。当与超分辨率受激发射损耗 (STED) 显微镜一起使用时，STED-FCS 可以测量活细胞中纳米级的扩散过程。在细胞质膜等二维 (2D) 系统中，环形耗尽焦点最常用于增加横向分辨率，导致观察体积减少超过 25 倍，达到超分子排列的相关规模. 然而，STED-FCS 在测量三维 (3D) 扩散时面临严重限制，这主要是由于激发焦点未耗尽区域的杂散背景贡献降低了信号质量并最终限制了分辨率。在本文中，我们研究了不同的 STED 限制模式如何缓解这个问题。通过模拟以及在溶液和细胞中使用荧光探针进行的实验，我们证明了双涡流相位掩模产生的相干混合 (CH) 耗尽模式最有效地减少了背景，从而在观察体积相当减少的情况下提供了卓越的信号质量. CH-STED 还具有对常见光学像差的最高鲁棒性，可被视为基于 STED-FCS 的可靠亚衍射尺度 3D 扩散研究的首选方法。我们证明了由双涡旋相位掩模创建的相干混合 (CH) 耗尽模式最有效地减少了背景，从而在观察体积的可比减少下提供了卓越的信号质量。CH-STED 还具有对常见光学像差的最高鲁棒性，可被视为基于 STED-FCS 的可靠亚衍射尺度 3D 扩散研究的首选方法。我们证明了由双涡旋相位掩模创建的相干混合 (CH) 耗尽模式最有效地减少了背景，从而在观察体积的可比减少下提供了卓越的信号质量。CH-STED 还具有对常见光学像差的最高鲁棒性，可被视为基于 STED-FCS 的可靠亚衍射尺度 3D 扩散研究的首选方法。