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    Label-free Mitotic Index

Highlights

Continuously monitor the mitotic index of a cell population in a non-invasive manner.

  • Robust segmentation and tracking of individual cells.
  • Time-resolved kinetic information related to behaviour of individual cells.
  • Automatically identify cells undergoing mitosis and extract the mitotic index.
  • Viable and unperturbed cells permit further downstream analysis.

Introduction

The mitotic index, or the percentage of a cell population that is undergoing mitosis, is an assay used commonly to determine the health of cells within a population. The mitotic index is often higher in cancerous cell populations due to increased cell proliferation. Current methods that exist to measure mitotic index, usually rely on the use of fluorescence markers and relatively high light levels, which can ultimately perturb the natural function of cells. This application note will demonstrate the capability of the Phasefocus™ system to image non-invasively and identify label free unique phase signatures that indicate when a single cell is undergoing mitosis. The nature of the continuous measurement, allows the mitotic index to be extracted at every time point, and subsequently a more robust and true representation of the behaviour of the cells to be categorised.

Methods

A mitotic event is clearly distinguishable using the Phasefocus Livecyte™ system. The event is easily identified as the cell adopts a more spherical morphology with a greater optical thickness than resting cells, causing a stronger phase shift and hence appearing brighter (Fig. 1). This is a fingerprint which is easily identifiable in the Phasefocus Cell Analysis Toolbox™ (CAT), and can be utilised to extract a suite of relevant information.

A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) Foetal bovine serum (FBS) and 1% (v/v) non-essential amino acids. For time-lapse experiments cells were plated in 8-well Lab-Tek II (Thermo Fisher Scientific Inc., Waltham, MA, USA) chamber slides and imaged every 5 minutes over a period of 24 hours. For measuring the mitotic index values of cell cultures, A549 cells were cultured as above and plated in coverslip-bottomed 35 mm dishes. Cells were treated with either 0 (control), 10, 25 or 50 ng/ml Nocodazole for a period of 6 hours to halt cells in the G2/M phase of the cell cycle. Large field-of-view images (750 x 750 µm) were then obtained of the cells.

Results

The images acquired from Livecyte exhibit an inherently low background intensity; the combination of this together with the high inherent contrast allows for robust segmentation and tracking of cells over time. Fig. 2 illustrates a single A549 cell which was automatically tracked over time up to the point of mitosis, after which the subsequent daughter cell was tracked up until the next mitotic event.

By plotting the mean cell optical thickness over time (Fig. 3), distinct peaks at the point of mitosis are clearly visible. We have previously reported that by processing the quantitative data from the images, the two populations can be presented numerically [1]. Here Nocodazole (Fig. 3) was used to manipulate the cell cycle of A549 cell cultures. Nocodozole acts to inhibit the microtubule formation at the G2/M stage of the cell cycle. A549 cells were treated with Nocodazole at a concentration of 10, 25 and 50 ng/ml for 6 hours. Using the Phasefocus system, a large field of view was acquired for the Nocodazole treated and control cells (Fig. 4).

The action of Nocodaozole can be seen visually by the greater proportion of cells held at mitosis, compared to the non-dividing population as the concentration of Nocodazole increases. Segmentation of the cells yields quantitative data for each individual cell. Plotting these data as a scatter plot of cell area against mean intensity the two populations become further apparent (Fig. 5). By taking a ratio of the two populations, (mitotic cells / non-mitotic cells) the mitotic index (MI) can be calculated. Following the 6hr treatment with 10, 25 and 50 ng/ml Nocodazole, the MI value also increased to 0.05, 0.23 and 0.36 respectively when compared to the control (MI= 0.03).

Figures

Label-free Mitotic Index

Figure 1. Phase images of A549 cells at various stages of mitosis.

Label-free Mitotic Index

Figure 2. A single A549 cell tracked over time starting at mitosis, up until mitosis of the daughter cell.

Label-free Mitotic Index

Figure 3. Mean cell thickness of a single A549 cell, tracked over time starting at mitosis and up until mitosis of the daughter cell.

Label-free Mitotic Index

Figure 4. Large field of view Phase image of A549 following a 6 hour treatment with 0 (control), 10, 25 and 50 ng/ml Nocodazole.

Label-free Mitotic Index

Figure 5. Scatter plots of cell area against mean intensity for each treatment, with the blue data points representing the cells at G2/M. Increasing the concentration of Nocodazole clearly shifts the cellular population towards the G2/M stage of the cell cycle.

References

1. Marrison, J., Räty, L., Marriott, P., and O’Toole, P., Ptychography – a label free, high-contrast imaging technique for live cells using quantitative phase information. Sci. Rep. 3, 2369 (2013).

Download the Application Note pdf

Conclusion

This application note clearly demonstrates the ability of the Phasefocus system to identify mitosis and measure the mitotic index label free. A key advantage of Livecyte is the non-toxic nature of its imaging modality, not only by virtue of the fact that fluorescent markers are not needed, but also because the laser illumination powers are very low (an order of magnitude lower than typically used in confocal microscopy). With this capability in mind the user can perform long (days to weeks) time-lapse studies of cell cultures, from which the mitotic index can be continuously computed. More importantly, during the course of a time-lapse measurement candidate drugs/treatments can be applied and their effects on the mitotic index can be evaluated. Furthermore, as cells are not altered by Livecyte, additional end point assays can be performed once the time-lapse imaging is complete. In conclusion, Livecyte offers a unique high contrast/quantitative label-free method for the analysis of mitosis and cell cycle dynamics.