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首页> 美国卫生研究院文献>other >Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)
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Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

机译：使用载物台和狭缝扫描来改善双视角倒置光片显微镜（diSPIM）的对比度和光学截面

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摘要

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.
机译：双向倒置选择性平面照明显微镜（diSPIM）可实现具有各向同性空间分辨率的高速，长期二维（4D）成像。它也与安装在玻璃盖玻片上的常规样品兼容。但是，在远离光束束腰的距离处加宽光片和样品引起的散射会降低diSPIM对比度和光学截面。我们描述了两个简单的改进，可以解决这两个问题，并且不需要对基本diSPIM进行其他硬件修改。首先，我们通过保持光片和检测光学器件固定，并通过固定的光片扫描样品（而不是像常规diSPIM那样通过固定的样品扫描加宽的光片和检测平面），展示了改进的diSPIM切片。这种阶段扫描方法允许在对横向扩展的样本（例如细胞单层中的固定微管或运动性线粒体）进行成像时使用更薄的薄片，并且比常规diSPIM产生更好的对比度。我们还使用了阶段扫描diSPIM来获得来自未压缩线虫胚胎的高质量4D核数据集，并进行谱系分析以跟踪97％的细胞直至抽搐。其次，我们通过使光片合成与科学互补金属氧化物半导体（sCMOS）检测器的滚动电子快门同步来描述厚散射样品的对比度提高。这种操作在检测路径上形成了一个虚拟的共聚焦狭缝，部分地消除了离焦的光线。我们通过成像活线虫胚胎中的花粉粒以及核和神经元结构来证明我们组合的阶段扫描和狭缝扫描方法的适用性。所有采集和分析代码均可在线免费获得。

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