Quantitative time-stretch imaging flow cytometry for high-throughput cell-cycle analysis

We present an image-based cell-cycle analysis at single-cell precision measured by a multimodal time-stretch imaging flow cytometer with a throughput >10,000 cells/sec. Flow cytometry is a potent tool for cellular phenotyping, which hold the keys to understand cellular functions [1]. However, it lacks the ability to detect and quantify biophysical phenotypes of cells, an effective intrinsic marker to probe a multitude of cellular processes. A notable example is cell growth, regarded as “one of the last big unsolved problems in cell biology” [2]. Although quantitative-phase microscopy (QPM) enables cell growth studies by quantifying the cell size and dry mass in a label-free manner, it has largely been restricted to adherent cell analysis with a low imaging throughput of ~100’s cells [3,4]. This limitation hampers high-throughput single-cell analysis which is now an unmet need in detection and analysis of rare metastatic cancer cells in a large population (thousands to even millions of cells) [3-6].

Leveraging its ultrafast frame rate, biophysical single-cell imaging based on time-stretch technology has shown its potential in scaling the imaging throughput by at least 2 orders of magnitude higher than the current techniques [7-9]. Here we present a further advancement by developing a multimodal time-stretch imaging flow cytometer for high-throughput image-based cell cycle analysis of cancer cells (metastatic breast cancer cell line, MDA-MB-231). The system features both QPM and fluorescence detection of individual suspended cells, flowing in a polydimethylsiloxane-based microfluidic channel at a high throughput of 10,000 cells/sec. Its configuration is similar to that reported in ref. [8-10] except an additional module for fluorescence excitation and detection. Fig. 1 shows some representative bright-field and quantitative-phase cell images captured by the system.

Not only can the system performs biophysical phenotyping inferred from the QPM, but also quantify the DNA content of single-cells with DNA-specific fluorescence labels. The combined information can be utilized for cell-growth monitoring and characterization of cell-cycle phases. The fluorescence signal is first used to identify the cell-cycle phase of individual cells and revealed the distribution of the cell-cycle phases in the whole population with G1/S/G2M phase, as 57.3%, 18.4% and 24.3% respectively (Inset of Fig. 2). Cell growth is then quantified by the cell dry mass which represents the protein content of each cell [11]. Throughout the cell-cycle, a progressive increase in dry mass from G1 (286±4 pg), via S (357±8 pg) to G2/M (438±11 pg) is observed (p <0.01) (Fig. 2). Furthermore, our analysis combining dry mass and fluorescence signal reveals that faster cell growth occurs in G1 and G2/M phases, in comparison to that during the S phase. It is consistent to the dominating action of DNA content duplication in this phase [12]. In summary, this integrated time-stretch imaging flow cytometer (QPM plus fluorescence detection) presents a powerful tool for large-scale single-cell analysis based on both molecular signatures (e.g. DNA content) and biophysical markers (e.g. dry mass) – opening a new paradigm in single-cell analysis of basic biology and new mechanistic insights into disease processes, not limited to cancer cell growth.