Quantifying of structures, kinetics and dynamics of necrobiological processes by image processed confocal fluorescence microscopy

Abstract

Translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet is presently discussed as a hallmark of apoptosis. The signalling which initialises the process and the translocation mechanism itself are still under investigation. Diffusion or enzymatic transport is suspected. Staurosporine induced PS-translocation was monitored in HCE-cells and in mouse lymphocytes (EL-4) by Annexin V-FITC (AV) fluorescence. QFM could not only reveal the percentage of AV positive and AV negative cells in both cultures, it could also derive that once initiated, PS-translocation in single HCE-cells is completed within less than ½h. This and other observations support nondiffusive PS-translocation. For toxicological investigations, early apoptotic parameters are essential. QFM detected two very early staurosporine induced effects in HCE- and EL-4-cells. In HCE-cells, a morphologic form of Karyorrhexis was discovered as early as 10min after induction. This form of chromatin condensation is probably initiated by the mitochondrial apoptotic inducing factor (AIF), which was recently discovered. Activated caspase-3, which is also discussed to initialise PS-translocation and chromatin condensation could be excluded for both events in HCE-cells. In EL-4-cells, perturbation of cellular migration and the exclusion of small cellular vesicles were detected within minutes after staurosporine induction. The results presented in this and other scientific publications validate the OFM method as a promising tool for future investigations. The method should work on many cellular events, in which fluorescent or non fluorescent texture or morphological features must be connected with statistical information