Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC.
Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC.
J Cell Biol. 2018 May 7;217(5):1869-1882. doi: 10.1083/jcb.201710087. Epub 2018 Feb 28.
Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.
荧光显微镜是一种强大的方法，可用于在高时空分辨率下研究亚细胞动力学；然而，传统的荧光显微镜技术强度大，会引入不必要的光损伤。光片荧光显微镜（LSFM）通过使用正交激发选择性地照亮检测物镜的焦平面来减轻这些问题。正交激发需要物理上限制检测物镜数值孔径（NA）的几何形状，从而限制了集光效率（亮度）和固有空间分辨率。我们提出了一种新的活细胞 LSFM 方法，即横向干涉倾斜激发（LITE），其中倾斜的光片照亮检测物镜的焦平面，而无需采用限制照明的方案。因此，LITE 与任何检测物镜兼容，包括油浸物镜，而不受 NA 上限的限制。LITE 将 LSFM 的低光损伤与高分辨率、高亮度以及基于盖玻片的物镜相结合。我们展示了 LITE 在高时空分辨率下对动物、真菌和植物模型生物进行数小时成像的实用性。
