Department of Neurosurgery, the First Medical Centre, Chinese People's Liberation Army General Hospital, Beijing 100853, China; Department of Neurosurgery, PLA (People's Liberation Army) Strategic Support Force Characteristic Medical Center, Beijing 100101, China.
Core Facility, Center of Biomedical Analysis, Tsinghua University, Beijing, 100091, China.
Neurosci Lett. 2020 Oct 15;737:135301. doi: 10.1016/j.neulet.2020.135301. Epub 2020 Aug 9.
To study the compatibility of traditional tracers and viral tracers with tissue clearing technology and to provide guidance in choosing suitable tracing methods for a specific tissue clearing technique.
Experiment 1: In this study, two different types of representative tracers, namely fluorescent dye tracers (Fluoro-Gold and Fluoro-Ruby) and viral tracers carrying fluorescent proteins (rAAV9-hSyn-mCherry-WPRE-pA and rAAV9-hSyn-EGFP-WPRE-pA), were selected to trace the cerebrospinal tract of the animals by microinjection. Furthermore, we presented the signal changes after using the three representative transparentizing methods, which included FRUIT (aqueous tissue clearing), 3DISCO (solvent-based tissue clearing), and uDISCO (solvent-based tissue clearing), were compared after slicing. Experiment 2: Based on the research mentioned above, Fluoro-Ruby was microinjected unilaterally into the primary motor cortex of rats, directly tracing the pyramidal tract to the spinal cord. Then, the entire brain and spinal cord were collected for tissue transparency using the 3DISCO method, after which three-dimensional imaging was performed using optical microscopic imaging equipment.
Experiment 1 indicated that Fluoro-Gold and Fluoro-Ruby displayed better compatibility with the three transparent methods. The viral tracer exhibited higher compatibility with the FRUIT method, while its compatibility with 3DISCO and uDISCO was low. Furthermore, GFP was quenched more quickly and seriously than cherry protein under the same experimental conditions. Experiment 2: The Fluoro-Ruby tag displayed the presence of long-distance axons. For microscopic imaging, light sheet microscopy and two-photon microscopy were both used to identify the signals of tracers in transparent tissue.
Both Fluoro-Gold and Fluoro-Ruby displayed excellent compatibility with tissue clearing technology, which, with dehydration and delipidation at its core, lead to quenching of fluorescence proteins, while exhibiting poor compatibility with viral tracers. In combination with tissue clearing technology and optical microscopy, the anterograde tracer Fluoro-Ruby could stereoscopically display the complete neural conduction pathway.
研究传统示踪剂和病毒示踪剂与组织透明化技术的兼容性，为特定组织透明化技术选择合适的示踪方法提供指导。
实验 1：本研究选择了两种不同类型的代表性示踪剂，即荧光染料示踪剂（Fluoro-Gold 和 Fluoro-Ruby）和携带荧光蛋白的病毒示踪剂（rAAV9-hSyn-mCherry-WPRE-pA 和 rAAV9-hSyn-EGFP-WPRE-pA），通过微注射将其标记动物的脑脊液轨迹。此外，我们展示了使用三种代表性透明化方法后的信号变化，包括 FRUIT（水性组织透明化）、3DISCO（溶剂型组织透明化）和 uDISCO（溶剂型组织透明化），并在切片后进行了比较。实验 2：基于上述研究，Fluoro-Ruby 被单侧注射到大鼠初级运动皮层，直接追踪到脊髓的锥体束。然后，使用 3DISCO 方法对整个大脑和脊髓进行组织透明化，然后使用光学显微镜成像设备进行三维成像。
实验 1 表明 Fluoro-Gold 和 Fluoro-Ruby 与三种透明方法具有更好的兼容性。病毒示踪剂与 FRUIT 方法的兼容性更高，而与 3DISCO 和 uDISCO 的兼容性较低。此外，在相同的实验条件下，GFP 的淬灭速度和程度均高于 cherry 蛋白。实验 2：Fluoro-Ruby 标签显示存在长距离轴突。对于显微镜成像，使用光片显微镜和双光子显微镜来识别透明组织中的示踪剂信号。
Fluoro-Gold 和 Fluoro-Ruby 均与组织透明化技术具有出色的兼容性，该技术以脱水和去脂化为核心，导致荧光蛋白淬灭，而与病毒示踪剂的兼容性较差。结合组织透明化技术和光学显微镜，顺行示踪剂 Fluoro-Ruby 可以立体显示完整的神经传导通路。
