Rgb Channels (in)dependence in Phase Contrast Microphotography

Time laps microscopy (microphotography) is one of the important measurements in many biological experiments. To describing life cycle of observed culture, cell division, fusion or comunication, both automaticaly or semiautomaticaly evaulation, is crucial to guarantee optimal camera settings, in case of observing long time experiment run by digital capture device. Monolayer of human cells – often vaguely referred as tissue culture – is the simplest approximation to the model of an organ from which the cells originate. From a small number of cells the culture is allowed to develop into monolayer and this process is observed under the microscope by a series of camera shots, confocal microscope fluorescence readouts etc. In this paper we describe the analysis of camera RGB channels (in)dependence of the most common system, i.e. HeLa cells observed by phase-contrast image enhancement. In a sense, this type of experiment is available to each medical practitioner or biotechnologist. We have chosen this setup as the most obvious example, yet we found out that even partial analysis of the experiment is sufficiently complicated to exploit the capacity limits of current computer technology. The phase contrast microscopy represents the observed object by the way of comparison of the refractivity index of the object (i.e. living cell) interior and refractivity index of free medium. As a result there is obtained image of light interference intensity in the focal point of the microscope. Refractivity index is a sum of non-absorptive, mainly electromagnetic, interactions of light with the matter. The objective assessment of the value of the refractivity index in the living cell interior is practically impossible using the phase contrast but was obtained by different methods. For the purpose of this discussion, it is also important that the refractivity index is wavelength dependent. If the detection system is for example a standard photograph, each of the three channels (Red, Green and Blue) carries different partly independent information which may be used for state definition. Therefore, the question of channel independence is important for any further analysis. Figure 1: Example of HeLa cells monolayer in phase contrast microscopy.