School of Biological Sciences, Washington State University, Pullman, WA, 99164, USA.
J Plant Physiol. 2020 Jan;244:153060. doi: 10.1016/j.jplph.2019.153060. Epub 2019 Nov 1.
The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.
将荧光探针微注射到活细胞中是现代细胞生物学工具包中的一个重要组成部分。微注射技术包括用微管穿透质膜，如果存在细胞壁，则用微管穿透细胞壁，并施加压力或电流将微管内容物驱动到细胞内。这些过程会干扰细胞功能，因此可能会产生假象。我们设计了扩散注射微管 (DIMP)，它避免了由于其流体内容物的体积大大减少以及利用扩散将货物递送到靶细胞而引起的大多数可能的假象。DIMP 已在植物、真菌和动物细胞中成功测试。使用 Nicotiana trichome 细胞刺穿后十分钟内细胞质动力学的连续性作为非侵入性的标准，我们发现 DIMP 比传统的压力微注射的破坏性要小得多。DIMP 的设计消除了其他微注射技术无法避免的主要假象来源。此外，DIMP 价格低廉，易于制作，并且可以在没有微操作器等特定设备的情况下应用。具有这些特点，DIMP 可能成为需要将材料以最小侵入性递送到活细胞的研究的首选工具。
