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Article

Optimization and Fabrication of Multi-Level Microchannels for Long-Term Imaging of Bacterial Growth and Expansion

1
Micro/Bio/Nanofluidics Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan
2
Department of Biomedical Engineering, Chang Gung University, Taoyuan 333, Taiwan
3
Biological Complexity Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan
*
Authors to whom correspondence should be addressed.
Academic Editors: Violeta Carvalho, Senhorinha Teixeira and João Eduardo P. Castro Ribeiro
Micromachines 2022, 13(4), 576; https://doi.org/10.3390/mi13040576
Received: 21 March 2022 / Revised: 4 April 2022 / Accepted: 5 April 2022 / Published: 7 April 2022
Bacteria are unicellular organisms whose length is usually around a few micrometers. Advances in microfabrication techniques have enabled the design and implementation of microdevices to confine and observe bacterial colony growth. Microstructures hosting the bacteria and microchannels for nutrient perfusion usually require separate microfabrication procedures due to different feature size requirements. This fact increases the complexity of device integration and assembly process. Furthermore, long-term imaging of bacterial dynamics over tens of hours requires stability in the microscope focusing mechanism to ensure less than one-micron drift in the focal axis. In this work, we design and fabricate an integrated multi-level, hydrodynamically-optimized microfluidic chip to study long-term Escherichia coli population dynamics in confined microchannels. Reliable long-term microscopy imaging and analysis has been limited by focus drifting and ghost effect, probably caused by the shear viscosity changes of aging microscopy immersion oil. By selecting a microscopy immersion oil with the most stable viscosity, we demonstrate successful captures of focally stable time-lapse bacterial images for ≥72 h. Our fabrication and imaging methodology should be applicable to other single-cell studies requiring long-term imaging. View Full-Text
Keywords: multi-level microfluidic device; live cell imaging; long-term microscopy imaging; focus drifting; immersion oil viscosity; bacterial population dynamics; single-cell studies; E. coli; mother machine; computational fluid dynamics multi-level microfluidic device; live cell imaging; long-term microscopy imaging; focus drifting; immersion oil viscosity; bacterial population dynamics; single-cell studies; E. coli; mother machine; computational fluid dynamics
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MDPI and ACS Style

Tsai, H.-F.; Carlson, D.W.; Koldaeva, A.; Pigolotti, S.; Shen, A.Q. Optimization and Fabrication of Multi-Level Microchannels for Long-Term Imaging of Bacterial Growth and Expansion. Micromachines 2022, 13, 576. https://doi.org/10.3390/mi13040576

AMA Style

Tsai H-F, Carlson DW, Koldaeva A, Pigolotti S, Shen AQ. Optimization and Fabrication of Multi-Level Microchannels for Long-Term Imaging of Bacterial Growth and Expansion. Micromachines. 2022; 13(4):576. https://doi.org/10.3390/mi13040576

Chicago/Turabian Style

Tsai, Hsieh-Fu, Daniel W. Carlson, Anzhelika Koldaeva, Simone Pigolotti, and Amy Q. Shen. 2022. "Optimization and Fabrication of Multi-Level Microchannels for Long-Term Imaging of Bacterial Growth and Expansion" Micromachines 13, no. 4: 576. https://doi.org/10.3390/mi13040576

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