Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

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Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device. / Schlimpert, Susan; Flärdh, Klas; Buttner, Mark.

I: Journal of Visualized Experiments, Nr. 108, 2016.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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T1 - Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device.

AU - Schlimpert, Susan

AU - Flärdh, Klas

AU - Buttner, Mark

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

U2 - 10.3791/53863

DO - 10.3791/53863

M3 - Article

C2 - 26967231

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 108

ER -