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

Area of Science:

• Biophysics
• Cell Biology
• Cytometry

Background:

• Cellular biophysical properties offer label-free phenotypes for cell characterization.
• Current methods lack throughput and reproducibility for large-scale single-cell analysis.
• Widespread adoption in cytometry is hindered by these limitations.

Purpose of the Study:

• To develop a high-throughput, label-free imaging flow cytometer for single-cell biophysical phenotyping.
• To address the limitations of existing quantitative phase imaging (QPI) techniques.
• To enable robust, large-scale single-cell characterization for biological discovery and diagnostics.

Main Methods:

• Development of a label-free imaging flow cytometer utilizing ultrafast quantitative phase imaging (QPI) termed multi-ATOM.
• Implementation of robust system calibration and validation for image acquisition and phenotyping reproducibility.
• Characterization of over 1,000,000 cells to establish high-dimensional single-cell biophysical phenotypic profiles.

Main Results:

• The multi-ATOM system achieves a throughput of >10,000 cells/s, enabling unprecedented label-free single-cell QPI.
• Demonstrated ability to generate high-dimensional biophysical phenotypic profiles at ultra-large-scale.
• Achieved high accuracy (~92-97%) in classifying multiple human leukemic cell types using label-free phenotypic data.

Conclusions:

• The developed multi-ATOM QPI flow cytometer overcomes throughput and reproducibility challenges in biophysical phenotyping.
• This technology enables high-statistical-power, label-free single-cell analysis for biological discovery.
• It holds significant potential for cost-effective clinical diagnostics and advancing mainstream cytometry.