Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Nat Methods. 2010 Aug;7(8):637-42. doi: 10.1038/nmeth.1476. Epub 2010 Jul 4.
Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo.
记录大的非透明样本（例如，正在发育的多细胞生物）的光镜图像，由于光散射会导致对比度降低，从而变得复杂。早期斑马鱼的发育可以通过标准的光片显微镜进行捕获，但需要新的成像策略来获得高质量的后期发育或透明度较低的生物体的数据。我们将数字扫描激光光片荧光显微镜与非相干结构照明显微镜（DSLM-SI）相结合，并创建了具有连续可调频率的结构照明模式。我们的方法可以将与样本相关的散射背景与信号荧光区分开来，从而去除离焦光并优化聚焦结构的对比度。DSLM-SI 提供了对照明模式的快速控制、出色的成像质量和高成像速度。我们对斑马鱼的发育进行了长达 58 小时的长期成像，并对早期黑腹果蝇的发育进行了快速的多视图成像。我们从果蝇 DSLM-SI 数据中重建了细胞位置，并创建了一个果蝇数字胚胎。
