Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.
Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.
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DOI：
10.1364/optica.3.000897
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发表时间：
2016-08-20
期刊：
影响因子：
10.4
通讯作者：
Shroff H
中科院分区：
文献类型：
--
作者：
Wu Y;Chandris P;Winter PW;Kim EY;Jaumouillé V;Kumar A;Guo M;Leung JM;Smith C;Rey-Suarez I;Liu H;Waterman CM;Ramamurthi KS;La Riviere PJ;Shroff H
大多数荧光显微镜效率低下，在任何时刻都只能收集一小部分发射光。除了浪费有价值的信号之外，这种低效率还降低了空间分辨率，并导致成像体积表现出显著的分辨率各向异性。我们描述了显微镜和计算技术，同时捕捉，随后融合和去卷积多个标本的意见，解决这些问题。与以前的方法，连续捕获多个视图，我们的方法提高了空间分辨率，而不引入任何额外的照明剂量或损害相对于传统成像的时间分辨率。当将我们的方法应用于单视图宽场或双视图光片显微镜时，我们实现了体积分辨率的两倍提高（~235 nm × 235 nm × 340 nm），如在各种样品上所证明的，包括弓形虫中的微管，芽孢杆菌中的SpoVM，以及真核细胞中的多种蛋白质分布和细胞器。在每种情况下，空间分辨率都通过利用以前未使用的荧光而得到了毫无缺点的提高。
Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.