Slabodnick Mark, Prevo Bram, Gross Peter, Sheung Janet, Marshall Wallace
Physiology Course, Marine Biological Laboratory.
J Vis Exp. 2013 Dec 19(82):e50848. doi: 10.3791/50848.
Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a "double decker" microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.
虽然伤口愈合通常是在整个组织层面进行研究的，但在许多情况下，单个细胞自身就能愈合伤口，在细胞内容物泄漏之前修复破损的细胞膜。巨大的单细胞生物天蓝喇叭虫，其细胞大小可达一毫米以上，一直是研究单细胞伤口愈合的经典模式生物。喇叭虫细胞被切成两半后仍能存活，甚至可以切割后再拼接在一起。但这种对切割的高耐受性引发了一个问题，即为什么细胞质不会简单地从切割处流出。在此，我们提出一种切割喇叭虫细胞的方法，同时以高空间和时间分辨率对切割附近细胞质的运动进行成像。我们方法的关键是使用“双层”显微镜配置，即在解剖显微镜下进行手术，该显微镜聚焦于一个腔室，同时使用带有高数值孔径镜头的倒置显微镜从下方进行高分辨率观察。这种设置在允许对手术过程进行高度控制的同时，仍能对细胞质进行高分辨率跟踪。
