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Ultracompact 3D microfluidics for time-resolved structural biology.

ABSTRACT: To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s^-1, which allows for the use of megahertz XFEL repetition rates. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering.

SUBMITTER: Knoska J

PROVIDER: S-EPMC6994545 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Ultracompact 3D microfluidics for time-resolved structural biology.

Knoška Juraj J Adriano Luigi L Awel Salah S Beyerlein Kenneth R KR Yefanov Oleksandr O Oberthuer Dominik D Peña Murillo Gisel E GE Roth Nils N Sarrou Iosifina I Villanueva-Perez Pablo P Wiedorn Max O MO Wilde Fabian F Bajt Saša S Chapman Henry N HN Heymann Michael M

Nature communications 20200131 1

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s<sup>-1</sup>, which allows for the use of megahertz XFEL repetition rates. By integrating an additional ...[more]

PMID: 32005876

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Project description:BackgroundDouble-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as Saccharomyces cerevisiae have been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair.ResultsWe used engineered S. cerevisiae cells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80-150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair.ConclusionsOur analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.

Dataset Information

Ultracompact 3D microfluidics for time-resolved structural biology.

Publications

Ultracompact 3D microfluidics for time-resolved structural biology.

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