Ryan Duncan P, Gould Elizabeth A, Seedorf Gregory J, Masihzadeh Omid, Abman Steven H, Vijayaraghavan Sukumar, Macklin Wendy B, Restrepo Diego, Shepherd Douglas P
Department of Physics, Colorado State University, Fort Collins, CO, 80523, USA.
Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO,, 80045, USA.
Nat Commun. 2017 Sep 20;8(1):612. doi: 10.1038/s41467-017-00514-7.
Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.
光学组织透明化技术彻底改变了研究人员对完整组织内分子、细胞和结构进行荧光测量的能力。所有光学透明化组织都存在一个常见的并发症，即空间异质的折射率，这会导致光散射和一阶散焦。我们设计了C-DSLM（透明组织数字扫描光片显微镜）作为一种低成本方法，旨在自动生成透明组织的聚焦图像。我们通过使用折射率匹配和不匹配的显微镜物镜对多个动物模型的组织中的荧光特征进行量化，展示了C-DSLM的灵活性和强大功能。这包括使用内源性荧光报告分子对完整组织内的髓磷脂轨迹进行独特测量，而典型的透明化方法使此类结构难以成像。对于所有测量，我们使用标准的连续组织切片和量化方法进行独立验证。与体积图像处理的进展相结合，C-DSLM提供了一种强大的方法来量化大组织切片内的亚微米特征。由于光散射显著减少，组织的光学透明化使得光学成像能够更深入组织。在此，瑞安等人解决了一阶散焦问题，这是一种非均匀折射率的假象，将光片显微镜扩展到部分透明的样本。
