利用斜平面结构照明实现光片显微镜的分辨率倍增。

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

机构信息

Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Cecil H. and Ida Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

出版信息

Nat Methods. 2022 Nov;19(11):1419-1426. doi: 10.1038/s41592-022-01635-8. Epub 2022 Oct 24.

Abstract

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

摘要

结构光照明显微镜(SIM)可在不要求高激光功率或专用荧光染料的情况下将荧光显微镜的空间分辨率提高一倍。然而,离焦荧光的激发会加速荧光漂白和光毒性。相比之下,光片荧光显微镜(LSFM)在很大程度上避免了离焦荧光的激发,从而能够以低荧光漂白和固有光学切片进行体积成像。将 SIM 与 LSFM 相结合,将能够以提高的分辨率进行温和的三维(3D)成像。然而,SIM 中实现各向同性分辨率加倍所需的照明图案的多个方向在光片格式中难以实现。在这里,我们展示了多维结构照明可以在倾斜平面显微镜中实现,这是一种使用单个物镜进行激发和检测的 LSFM 技术,其实现方式非常简单。我们证明了横向各向同性分辨率低于 150nm,与传统 SIM 系统相比,光毒性更低,并且体积采集速度超过 1Hz。

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