Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, 20892, Maryland, USA.
Department of Radiology, University of Chicago, Chicago, 60637, Illinois, USA.
Nat Commun. 2017 Nov 13;8(1):1452. doi: 10.1038/s41467-017-01250-8.
Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.
光片荧光显微镜(LSFM)能够实现高速、高分辨率和对活标本进行长时间的温和成像。在这里,我们描述了一种无需修改基础显微镜即可提高 LSFM 的时空分辨率和采集效率的技术。通过在反射盖玻片上对样本成像,我们能够在 250ms 内同时采集四个互补视图,使速度提高一倍,并与对称双视图 LSFM 相比提高了信息量。我们还报告了一种改进的去卷积算法,该算法可去除相关的荧光背景污染并融合所有视图以进行分辨率恢复。此外,我们通过将该方法应用于单视图 LSFM 来提高空间分辨率(在所有三个维度上均小于 300nm),从而可以同时获取两个高分辨率视图,否则由于高数值孔径下的空间限制,很难获得这两个视图。我们在各种样本中展示了我们方法的广泛适用性,研究了细胞中线粒体、膜、高尔基体和微管的动力学以及线虫胚胎中的钙活性。
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