Phase Holographic Imaging


Microscopy Techniques

Cell tracking

Without stains or labels, the HoloMonitor cytometers automatically track adherent cell movement, speed and morphology changes over time


Overlay of chemotaxis cell tracks created by the HoloMonitor from Phase Holographic Imaging


Plot of chemotaxis cell tracks created by the HoloMonitor from Phase Holographic Imaging


Figure 1: (a) Time-lapse video. Cells are artificially colored to show the optical thickness. Purple cells are optically thicker. (b) Overlay of cell tracks. Lighter cells are opti­cally thicker. (c) Spatial plot of cell move­ment.

The HT-1080 cells were grown in a gel matrix on a μ-Slide Chemotaxis 3D slide from IBIDI. The cells were imaged once every 5 minutes for 16 hours. Bovine serum was used to stimulate cell motion.

Time-lapse imaging is commonly used to track cell movement when studying cell motility, conducting chemotaxis experiments or when using cell migration assays. Traditionally, cells need to be stained/labeled to allow automated cell tracking. However, the staining is invasive and may change cell behavior or even kill cells prematurely. Cells are therefore preferable tracked without the use of stains or labels.

State of the art

Conventional phase contrast microscope techniques give unstained cells contrast by inducing image artifacts. These artifacts make the image visually pleasing. But they distort cell morphology and make automated image processing challenging. When using conventional microscopy techniques, label-free cell tracking therefore requires that each cell is manually identified in each image frame, involving several thousand mouse clicks over many hours in front of the computer.

Automated tracking

The HoloMonitor records images using novel quantitative phase contrast techniques. Such techniques measure the true optical thickness of a cell in each image point. This allow Holo­Monitor users to effortlessly track the movement of unstained cells. But, it also allow cell volume and morphology to be tracked over time as well.

In figure 1a, a time-lapse video recorded by the HoloMonitor is shown. By using the HoloMonitor tracking software, selected cells were automatically tracked to give the cell tracking data shown in figure 1b and c.

How it is done

Video showing how to track cells using the HoloMonitor software.

  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 cells are correctly identified and outlined.
  4. Selected the Cell Tracking tab and track cells as shown in the video.
  5. Optionally select the Export Images tab to create a time-lapse video.

Download demo software

By Peter Egelberg