Kumar Abhishek, Wu Yicong, Christensen Ryan, Chandris Panagiotis, Gandler William, McCreedy Evan, Bokinsky Alexandra, Colón-Ramos Daniel A, Bao Zhirong, McAuliffe Matthew, Rondeau Gary, Shroff Hari
1] Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Program in Cellular Neuroscience, Neurodegeneration and Repair, Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA.
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Nat Protoc. 2014 Nov;9(11):2555-73. doi: 10.1038/nprot.2014.172. Epub 2014 Oct 9.
We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ∼6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3-8 h, depending on the size of the data.
我们描述了一种紧凑型双视角倒置选择性平面照明显微镜（diSPIM）的构建和使用方法，用于在亚细胞分辨率下对活体样本进行延时体积（4D）成像。我们的方案使具有一定显微镜经验的生物学家能够利用市售部件组装diSPIM，进行光学元件校准和测试系统性能，制备样本，并通过我们的软件控制硬件和数据处理。与现有的光片显微镜方案不同，我们的方法不需要将样本嵌入琼脂糖中；相反，样本是在常规玻璃盖玻片上制备的。本方案以组织培养细胞和秀丽隐杆线虫胚胎为例；成功实施该方案可在这些样本上实现各向同性分辨率和高达每秒几个体积的采集速度。组装和验证diSPIM性能大约需要6天，样本制备和数据采集最多需要5天，后处理需要3 - 8小时，具体取决于数据大小。
