Trypanosoma brucei, the causative agent of human African trypanosomiasis, employs a flagellum for dissemination within the parasite’s mammalian and insect hosts. T. brucei cells are highly motile in culture and must be able to move in all three dimensions for reliable cell division. These characteristics have made long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of a variety of important cell-cycle events. To address this issue, we have devised an imaging approach that confines cells to small volumes that can be imaged continuously for up to 24 h. This system employs cast agarose microwells generated using a PDMS stamp that can be made with different dimensions to maximize cell viability and imaging quality. Using this approach, we have imaged individual T. brucei through multiple rounds of cell division with high spatial and temporal resolution. We have employed this method to study the differential rate of T. brucei daughter cell division and show that the approach is compatible with loss-of-function experiments such as small molecule inhibition and RNAi. We have also developed a strategy that employs in-well “sentinel” cells to monitor potential toxicity due to imaging. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.

中文翻译：

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用长期锥虫活细胞成像揭示时空动态

布氏锥虫是人类非洲锥虫病的病原体，它利用鞭毛在寄生虫的哺乳动物和昆虫宿主内传播。T. brucei细胞在培养中具有高度运动性，并且必须能够在所有三个维度上移动才能进行可靠的细胞分裂。这些特征使得活体布氏锥虫的长期显微成像细胞具有挑战性，这限制了我们对各种重要细胞周期事件的理解。为了解决这个问题，我们设计了一种成像方法，将细胞限制在可以连续成像长达 24 小时的小体积内。该系统采用使用 PDMS 印章生成的浇铸琼脂糖微孔，该印章可以制成不同的尺寸，以最大限度地提高细胞活力和成像质量。使用这种方法，我们通过具有高空间和时间分辨率的多轮细胞分裂对单个布氏锥虫进行了成像。我们采用这种方法来研究布氏锥虫的差异率子细胞分裂并表明该方法与功能丧失实验兼容，例如小分子抑制和 RNAi。我们还开发了一种策略，使用井内“哨兵”细胞来监测成像引起的潜在毒性。这种活细胞成像方法将为研究以前无法进入的锥虫中的各种细胞事件提供一种新的途径。