Mateescu, B. | Search bioRxiv

bioRxiv2019

Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection

Flow cytometry is widely recognized as the gold-standard technique for the analysis and enumeration of heterogeneous cellular populations and has become an indispensable tool in diagnostics,1 rare-cell detection2 and single-cell proteomics.3 Although contemporary flow cytometers are able to analyse many thousands of cells per second, with classification based on scattering or fluorescence criteria, the vast majority require unacceptably large sample volumes, and do not allow the acquisition of spatial information. Herein, we report a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence and brightfield detection at analytical throughputs in excess of 60,000 cells/s and 400,000 cells per second respectively. Our imaging platform is capable of multi-parametric fluorescence quantification and subcellular (co-)localization analysis of cellular structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of our approach by performing challenging high-throughput localization analysis of cytoplasmic RNA granules in yeast and human cells. Results suggest significant utility of the imaging flow cytometer in the screening of rare events at the subcellular level for diagnostic applications.

bioengineering↗

bioRxiv2019

Oscillatory Viscoelastic Microfluidics for Efficient Focusing and Separation of Nanoscale Species

The ability to precisely control particle migration within microfluidic systems is essential for focusing, separating, counting and detecting a wide range of biological species. To date, viscoelastic microfluidic systems have primarily been applied to the focusing, separation and isolation of micron-sized species, with their use in nanoparticle manipulations being underdeveloped and underexplored, due to issues related to nanoparticle diffusivity and a need for extended channel lengths. To overcome such issues, we herein present sheathless oscillatory viscoelastic microfluidics as a method for focusing and separating both micron and sub-micron species. To highlight the efficacy of our approach, we segment our study into three size regimes, namely micron (where characteristic particle dimensions are above 1 m), sub-micron (where characteristic dimensions are between 1 m and 100 nm) and nano (where characteristic dimensions are below 100 nm) regimes. Based on the ability to successfully manipulate particles in all these regimes, we demonstrate the successful isolation of p-bodies from biofluids (in the micron regime), the focusing of {lambda}-DNA (in the sub-micron regime) and the focusing of extracellular vesicles (in the nano-regime). Finally, we characterize the physics underlying viscoelastic microflows using a dimensionless number that relates the lateral velocity (due to elastic effects) to the diffusion constant of the species within the viscoelastic carrier fluid. Based on the ability to precisely manipulate species in all three regimes, we expect that sheathless oscillatory viscoelastic microfluidics will provide for significant new opportunities in a range of biological and life science applications.

bioengineering↗