Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM).
Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM).
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DOI：
10.1086/689589
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发表时间：
2016-08
期刊：
影响因子：
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通讯作者：
Shroff H
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文献类型：
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作者：
Kumar A;Christensen R;Guo M;Chandris P;Duncan W;Wu Y;Santella A;Moyle M;Winter PW;Colón-Ramos D;Bao Z;Shroff H
双视图倒置选择性平面照明显微镜（diSPIM）能够实现具有各向同性空间分辨率的高速，长期，四维（4D）成像。它也与传统的玻璃盖玻片上的样品安装兼容。然而，在远离束腰的距离处的光片的加宽和样品引起的散射降低了diSPIM对比度和光学切片。我们描述了两个简单的改进，解决这两个问题，不需要额外的硬件修改的基础diSPIM。首先，我们通过保持光片和检测光学器件静止，并通过静止光片扫描样品（而不是如在常规diSPIM中那样通过静止样品扫描加宽光片和检测平面）来证明改进的diSPIM切片。这种阶段扫描方法允许在成像横向延伸的样品时使用更薄的片材，例如固定的微管或细胞单层中的运动线粒体，并且产生比传统的diSPIM更精细的对比度。我们还使用阶段扫描diSPIM来获得来自未压缩的线虫胚胎的高质量4D核数据集，并进行谱系分析以跟踪97%的细胞直到抽搐。其次，我们描述了厚，散射标本的对比度的改善同步光片合成与滚动，我们的科学互补金属氧化物半导体（sCMOS）探测器的电子快门。这种机动在检测路径中形成虚拟共焦狭缝，部分地去除失焦光。我们证明了我们的相结合的阶段和狭缝扫描方法的适用性成像花粉粒和核和神经元结构在活线虫胚胎。所有的采集和分析代码都可以在网上免费获得。
Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, fourdimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning-methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.