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A time-lapse image sequence of an unstained DU145 cell undergoing apoptosis induced by etoposide. The images were captured using a HoloMonitor time-lapse cytometer.
In the line profile of a traditional phase-contrast image, the background value cannot be accurately determined and a characteristic bright halo is seen around the edges of the cells. In this type of image, individual cells cannot be identified, i.e., cell segmentation is impossible.
In contrast, holographic phase-shift images can be quantified because they reflect the optical thickness of the cell and optical density variations in the specimen. Additionally, holographic images have a background level of zero and the intensities of events are measured as positive values.
HoloMonitor methodology enables reliable cell segmentation through the identification of individual cells, shown in the image by yellow cellular boundaries defined by a proprietary software algorithm.
Example of a HoloMonitor 3D phase-shift image of cells. The height of the cell and its color tone correspond to the phase shift, at a specific image point. The phase shift in turn is proportional to how much the light has slowed when passing through the cell.
The described principle of phase-shift imaging is the key to gathering quantitative data on cell characteristics. However, the true power of image cytometry first emerges when several images of the same cells are recorded over time. The HoloMonitor design utilizes recent technological advances to allow time-lapse image sequences of cultured cells to be effortlessly recorded over long time periods.
When a HoloMonitor® time-lapse cytometer is installed in a cell incubator, kinetic data are obtained using time-lapse imaging, i.e., serial images are acquired at regular time intervals to capture the dynamics of what is being observed. Images can be recorded at flexible intervals, down to 1 image/sec, depending on the application, and played back as a video recording at different speeds to aid analysis.
From recorded time-lapse sequences, the HoloMonitor software helps the user automatically extract individual cell data from data on a cell population. Individual cell data – for example cell count, cell morphology, cell velocity, and cell division rate – from a single time-lapse video recording can be used in a range of applications.
Time-lapse cytometry provides a new tool for studying and understanding cellular dynamics: