Quantification of the Time Course of Proliferation and Scattering Activity of Cells in Time Lapse Videos

Abstract

Introduction: Cell cultures frequently are observed using time lapse microscopic recording. Without the use of fluorescence markers the automatic quantification of morphological changes often fails. We established a semi automated technique allowing the quantification of the following parameters: number of cells and nuclei, cell death and division events and the extent of cell scattering. Errors caused by changing illumination conditions and scattering of frame positions have to be corrected.

Methods: Time lapse videos of rat hepatocytes cultured for 5d were used. The scattering of frame positions was corrected. The counting of objects and events of interest starts with the manual marking of all objects in the first image. In subsequent images only changes are captured. Analysis of the markers is done automatically. The scattering activity is quantified using a cross-correlation based algorithm comparing every image with its successor. Errors are filtered by an appropriate curve smoothing method.

Results: Several cell culture videos were processed and analyzed. The time course of the relative population of nuclei of vital and dead cells was displayed graphically as a step function and relevant events were shown as impulses. The scattering activities based on the correlation coefficient were calculated and - due to a lack of methods for validation - compared to the subjective judgment of the scattering by experienced experimenters.

Discussion and Conclusions: The semi automated method allows the quantification of cell death and cell division events in time lapse videos of cell cultures. Hence the assessment of the approximate time of events is possible. The new method to quantify the cell’s scattering activity proved to be robust enough against errors and sensitive to the scattering. The results compare very well to the subjective judgment of the observed scattering. The combination of both methods allows new insights into the development of a cell culture.