Quantitative Phase Imaging Flow Cytometry for Ultra-Large-Scale Single-Cell Biophysical Phenotyping

Cytometry A. 2019 May;95(5):510-520. doi: 10.1002/cyto.a.23765. Epub 2019 Apr 22.

Abstract

Cellular biophysical properties are the effective label-free phenotypes indicative of differences in cell types, states, and functions. However, current biophysical phenotyping methods largely lack the throughput and specificity required in the majority of cell-based assays that involve large-scale single-cell characterization for inquiring the inherently complex heterogeneity in many biological systems. Further confounded by the lack of reported robust reproducibility and quality control, widespread adoption of single-cell biophysical phenotyping in mainstream cytometry remains elusive. To address this challenge, here we present a label-free imaging flow cytometer built upon a recently developed ultrafast quantitative phase imaging (QPI) technique, coined multi-ATOM, that enables label-free single-cell QPI, from which a multitude of subcellularly resolvable biophysical phenotypes can be parametrized, at an experimentally recorded throughput of >10,000 cells/s-a capability that is otherwise inaccessible in current QPI. With the aim to translate multi-ATOM into mainstream cytometry, we report robust system calibration and validation (from image acquisition to phenotyping reproducibility) and thus demonstrate its ability to establish high-dimensional single-cell biophysical phenotypic profiles at ultra-large-scale (>1,000,000 cells). Such a combination of throughput and content offers sufficiently high label-free statistical power to classify multiple human leukemic cell types at high accuracy (~92-97%). This system could substantiate the significance of high-throughput QPI flow cytometry in enabling next frontier in large-scale image-derived single-cell analysis applied in biological discovery and cost-effective clinical diagnostics. © 2019 International Society for Advancement of Cytometry.

Keywords: imaging flow cytometry; label-free biophysical phenotyping; quantitative phase imaging; ultrafast single cell imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biophysical Phenomena*
  • Blood Cells / pathology
  • Calibration
  • Cell Line, Tumor
  • Flow Cytometry / methods*
  • Humans
  • Image Processing, Computer-Assisted*
  • Leukemia / pathology
  • Multivariate Analysis
  • Phenotype
  • Reproducibility of Results
  • Single-Cell Analysis*