Phase Holographic Imaging

HoloMonitor

Microscopy Techniques

Cell cycle

The HoloMonitor time-lapse cytometers provide powerful, non-invasive, assays for cell mitosis and cell cycle analysis

Figure 1: Time-lapse recording of dividing L929 cells.

Figure 2: Selected time-lapse images of dividing PanC-1 cells. The cells were imaged every 5 minutes for 72 hours, using the HoloMonitor M3.

Capturing the cell cycle

Even though poorly studied, it is well known that cell morphology reflects the cell cycle phases (Porter et al K. Porter, D. Prescott and J. Frye
'CHANGES IN SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS DURING THE CELL CYCLE',
The Journal of Cell Biology, vol. 57, no. 3, pp. 815–836, 1973
). The morphological and cytometrical changes during the cell cycle phases are captured using the sequential imaging process provided by the HoloMonitor. The auto focusing capabilities combined with the non-phototoxic imaging make long-term time-lapse studies of cell cultures simple and straight forward. In such studies (figure 1), cell division can be analyzed step by step (figure 2):

  1. The typical rounding up of the cell as it organizes its chromatin,
  2. the elongation of the cell with two separate chromatin bars,
  3. the separation of the two daughter cells and
  4. finally the spreading out of the new cells to their flat shape with asymmetrical protrusions which can all be traced and analyzed.

Analyzing the cell cycle

Besides creating fascinating time-lapse videos, the robust image segmentation make it possible to create scatter plots and histograms, displaying parameters of choice over time, e.g. cell volume, optical thickness or adherent surface area. The huge amount of data recorded for each cell and for the whole cell population can either be processed by the HoloMonitor software or exported to Microsoft Excel/OpenOffice.

How it is done

  1. Place the cell culture on the HoloMonitor stage.
  2. Selected the Live Capture tab in the Holo­Monitor software.
    1. If necessary, adjust the focus.
    2. Enter the time-lapse duration and the time interval between each image frame.
    3. Start image acquisition.
  3. After the time-lapse sequence has been recoded, select the Identify Cells tab.
    1. Adjust the two settings so that the cells are correctly identified and outlined.
  4. Selected the Cell Tracking tab and track cells as shown in the video below.
  5. Optionally select the Export Images tab to create a time-lapse video.

Download demo software

Video showing how to track cells using the HoloMonitor software.

Label-free cell cycle analysis

The time-plots below show how cell morphology typically change through the cell cycle. The cell volume
(
)
instantly drops after cell division. The cell volume remains constant for some time after cell division. It then gradually increases until just before the pre-mitosis contraction. During the mitotic contraction the optical thickness
(
)
dra­ma­tic­ally increase.

A rare hesitating cell division

Usually, cells follow the cell division cycle without reverting back to a previous cell cycle phase. In the left video clip a cell curl up to divide. Surprisingly the cell aborts the cell division process. The cell attempts and fails to divide several times. It is not until after the cell has interacted with its neighbors that it finally manages to divide successfully.

By Peter Egelberg