High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes

Gregor Holzner,Bogdan Mateescu,Daniel Van Leeuwen,Gea Cereghetti,Reinhard Dechant,Stavros Stavrakis,Andrew Demello

doi:10.1016/j.celrep.2021.108824

Gregor Holzner, Bogdan Mateescu + Show 5 more

Open Access

https://doi.org/10.1016/j.celrep.2021.108824

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Abstract

We present a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection at ultra-high analytical throughput. The imaging platform is capable of multiparametric fluorescence quantification and sub-cellular localization of these structures down to 500nm with microscopy image quality. We demonstrate the efficacy of the approach through the analysis and localization of P-bodies and stress granules in yeast and human cells using fluorescence and bright-field detection at analytical throughputs in excess of 60,000 and 400,000 cells/s, respectively. Results highlight the utility of our imaging flow cytometer in directly investigating phase-separated compartments within cellular environments and screening rare events at the sub-cellular level for a range of diagnostic applications.

Highlights

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 (Hasegawa et al, 2013), rare-cell detection (Boraldi et al, 2016), and single cell proteomics (Gauthier et al, 2008)
Microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence detection at ultra-high analytical throughput
The imaging platform is capable of multiparametric fluorescence quantification and sub-cellular localization of these structures down to 500 nm with microscopy image quality

Summary

Introduction

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 (Hasegawa et al, 2013), rare-cell detection (Boraldi et al, 2016), and single cell proteomics (Gauthier et al, 2008). Imaging flow cytometry (IFC) is a hybrid technology, incorporating the advantages of microscopy and flow cytometry, for high-throughput imaging of cells within flowing environments. Such an approach provides for enormous enhancements in information content but is accompanied by a number of technological challenges, including the need to acquire high-resolution (blur-free) images of single cells moving at high speed, the integration of multiple imaging modes (such as fluorescence, brightfield, and dark-field imaging) and the realization of adequate detection sensitivities when using short exposure times (Basiji et al, 2007). It should be noted that imaging cells in flow avoids the requirements of membrane staining, which is especially important for performing image segmentation under static conditions (Berchtold et al, 2018)

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