csiLSFM combines light- sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm
csiLSFM combines light- sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm
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DOI：
10.1073/pnas.1609278114
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发表时间：
2017-05-09
影响因子：
11.1
通讯作者：
Stelzer, Ernst H. K.
中科院分区：
文献类型：
--
作者：
Chang, Bo-Jui;Meza, Victor Didier Perez;Stelzer, Ernst H. K.
基于光片的荧光显微镜（LSFM）的特点是在激发过程中进行光学切片。它最大限度地减少了荧光团漂白以及光毒性效应，并提供了真正的轴向分辨率。检测路径类似于常规荧光显微镜的特性。结构照明显微镜（SIM）是有吸引力的超分辨率，因为它的中等激发强度，高采集速度，并与所有的荧光团兼容。我们将SIM引入LSFM，因为该组合将横向分辨率推到线性SIM的物理极限。该仪器需要三个物镜，并依赖于控制两个反向传播相干光片的方法，这两个相干光片在检测透镜的焦平面中产生激发图案。在远场中具有最细线间距的SIM图案沿沿着多个取向变得可用。灵活的控制旋转，频率和相移的完美调制的光片被证明。珠粒的图像证明了亚100 nm的近各向同性横向分辨率。酵母内质网的图像显示，相干结构照明（csi）LSFMperforms与生理相关标本。
Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFMperforms with physiologically relevant specimens.