Department of Biological Sciences, Purdue University.
Bindley Bioscience Center, Purdue University.
J Vis Exp. 2024 Sep 27(211). doi: 10.3791/67313.
Zebrafish embryos are transparent and thus uniquely suited for noninvasive intravital imaging of fundamental processes, such as wound healing and immune cell migration. Microfluidic devices are used for entrapment to support long-term imaging of multicellular organisms, including zebrafish. However, the fabrication of these devices using soft lithography requires specialized facilities and competency in 3D printing, which may not be accessible to every lab. Our adaptation of a previously developed low-cost polyethylene terephthalate lamination method for constructing microfluidic devices increases accessibility by enabling design fabrication and iteration for a fraction of the technical investment of conventional techniques. We use a device made with this method, the Rotational Assistant for Danio Imaging of Subsequent Healing (RADISH), to accommodate drug treatment, manual wounding, and long-term imaging of up to four embryos in the same field of view. With this new design, we successfully capture gross morphological characteristics of the calcium signal around laser ablation and manual transection wounds for multiple embryos in the 2 h immediately following injury, as well as neutrophil recruitment to the wound edge for 24 h.
斑马鱼胚胎是透明的，因此非常适合非侵入性活体成像，可用于研究伤口愈合和免疫细胞迁移等基本过程。微流控设备可用于捕获以支持包括斑马鱼在内的多细胞生物的长期成像。然而，使用软光刻制造这些设备需要专门的设施和 3D 打印方面的专业知识，而这可能不是每个实验室都具备的。我们对以前开发的低成本聚对苯二甲酸乙二醇酯层压方法进行了改进，用于构建微流控设备，通过降低技术投资，增加了设计、制造和迭代的可及性，而这仅为传统技术的一小部分。我们使用这种方法制造的设备，即 Rotational Assistant for Danio Imaging of Subsequent Healing（RADISH），可进行药物处理、手动造伤以及在同一视场中对多达四个胚胎进行长期成像。使用这个新设计，我们能够成功捕获激光消融和手动横切伤后 2 小时内多个胚胎周围钙信号的大体形态特征，以及中性粒细胞向伤口边缘的募集，持续 24 小时。
