Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.
Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.
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DOI：
10.3791/53863
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发表时间：
2016-02-28
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影响因子：
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通讯作者：
Buttner J
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文献类型：
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作者：
Schlimpert S;Flärdh K;Buttner J
单细胞水平生物过程的活细胞成像有助于我们目前对细菌细胞亚细胞组织的理解。然而，由于技术上的困难，应用延时显微镜来研究产孢丝状细菌链霉菌发育的细胞生物学过程一直受到阻碍。在这里，我们提出了一个方案，以克服这些限制，通过培养新的模式物种，委内瑞拉链霉菌，在一个市售的微流控装置连接到一个倒置荧光宽视场显微镜。与经典的模式物种——色链霉菌不同，委内瑞拉葡萄球菌在液体中产孢子，这使得微流体生长室可以长期培养和显微镜下监测委内瑞拉葡萄球菌的细胞发育和分化。除了监测形态学变化，荧光标记的靶蛋白的时空分布也可以通过延时显微镜可视化。此外，微流控平台提供了交换培养基的实验灵活性，该培养基用于在微流控室中刺激委内瑞拉螺孢子的详细方案。委内瑞拉整个生命周期的图像以特定的间隔获得，并在开源软件Fiji中进行处理，以制作记录时间序列的电影。
Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.