According to Mie theory, variations in optical properties lead to angle-dependent intensity variations 14 which consequently suggest the angle-resolved detection. For example, object size information is contained in small-angle scattering while shape information is found at larger angles 9. Since different scattering processes (diffraction, refraction, and reflection) dominate the intensity of scattered light at different angles 9, measuring light scattering over multiple angles provides more detailed information about the size, shape, morphology, and refractive index of particles 10, 11 and cells 12, 13. The FSC correlates to the volume whereas the SSC signal correlates to the granular structure of the cell 8. The scattered light signals are commonly detected at two angular ranges of ~0вЂ“20В° (forward scattered light, FSC) and around 90В° (side scattered light, SSC) relative to the direction of the excitation laser beam. In addition to detecting fluorescence, a state-of-the-art flow cytometer measures the scattered light from cells. However, a flow-cytometric method to identify cell cycle stages without labeling is only available via elaborate analysis of imaging 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. However, the identification and isolation of primary cell populations according to their cell cycle stages is of prime concern to, e.g., avoid that different cell cycle stages of cells introduce variance in single-cell RNAseq data 3 that would require correction in the data analysis. 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. 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. This approach is technically compatible with conventional flow cytometers and paves the way for label-free cell sorting.ДЇlow cytometeryВ (FC) is a powerful tool to investigate the physical and chemical characteristics of single cells and corresponding fluorescence-activated cell sorting is used to extract cell populations for further downstream analysis, such as gene expression by RNAseq. Our results demonstrate that MAPS-FC can be used to determine cell properties that are otherwise only accessible by invasive labeling. The results were validated by DNA staining and by sorting and re-analysis of isolated G1, S, and G2/M populations. 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. 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. 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. However, analysis is often constrained by the availability of detection reagents or functional changes of cells caused by fluorescent staining. 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.

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