Multi-angle pulse shape detection of scattered light in flow cytometry for label-free cell cycle classification

Daniel Kage,Claudia Giesecke-Thiel,Toralf Kaiser,Kristen Feher,Jenny Kirsch,Kerstin Heinrich,Konrad V Volkmann

doi:10.1038/s42003-021-02664-3

Abstract

Flow cytometers are robust and ubiquitous tools of biomedical research, as they enable high-throughput fluorescence-based multi-parametric analysis and sorting of single cells. However, analysis is often constrained by the availability of detection reagents or functional changes of cells caused by fluorescent staining. Here, we introduce MAPS-FC (multi-angle pulse shape flow cytometry), an approach that measures angle- and time-resolved scattered light for high-throughput cell characterization to circumvent the constraints of conventional flow cytometry. In order to derive cell-specific properties from the acquired pulse shapes, we developed a data analysis procedure based on wavelet transform and k-means clustering. We analyzed cell cycle stages of Jurkat and HEK293 cells by MAPS-FC and were able to assign cells to the G1, S, and G2/M phases without the need for fluorescent labeling. The results were validated by DNA staining and by sorting and re-analysis of isolated G1, S, and G2/M populations. Our results demonstrate that MAPS-FC can be used to determine cell properties that are otherwise only accessible by invasive labeling. This approach is technically compatible with conventional flow cytometers and paves the way for label-free cell sorting.

Highlights

Flow cytometers are robust and ubiquitous tools of biomedical research, as they enable highthroughput fluorescence-based multi-parametric analysis and sorting of single cells
A flow-cytometric method to identify cell cycle stages without labeling is only available via elaborate analysis of imaging Flow cytometery (FC) data[4] which is currently limited in throughput[5], or Raman-based methods[6,7] that focus on chemical composition but not on optical properties
To determine the cell cycle phases of HEK293 cells, cells were stained with propidium iodide (PI) and BrdU and measured with MAPS-FC while performing conventional fluorescence detection (Fig. 1a)

Summary

Introduction

Flow cytometers are robust and ubiquitous tools of biomedical research, as they enable highthroughput fluorescence-based multi-parametric analysis and sorting of single cells. Flow-cytometric characterization is mainly based on the measurement of the intensities of scattered light and fluorescence light emitted from cells or particles after fluorescent labeling For some purposes, such as cell cycle phase analysis, detrimental manipulation like fixation is required for DNA labeling, which restricts downstream experiments, or the fluorescent dyes are potentially toxic or interfere with cell functions[1,2] which may prevent the cultivation of cells after single-cell sorting. Even though standard instruments do acquire these intensity functions of time (pulse shapes (PS)), due to the technical setup, only three parameters (height H, area A, and width W) are derived from the PS in signal processing while the rest of the information is discarded These key parameters enable the differentiation of cell doublets or tracking protein aggregation[19], for example. To the best of our knowledge, a combination of both angular and temporal resolution has not yet been investigated

Methods

Results

Conclusion