Calibration and quantification of fast intracellular motion (FIM) in living cells using correlation analysis

Video rate confocal laser scanning microscopy at the highest spatial and temporal resolution of backscattered light (BSL) imaging allowed for regular observation of fast intracellular motion (FIM) first revealed in living neoplastic cells. However, the absence of an objective evaluation has hampered further study of the mechanisms and biological significance of FIM. Particularly, a quantification of apparent differences in velocities that would complement and improve the current demonstration of FIM by color coding using the combination of red-green-blue (RGB) images had been missing. Standard methods of tracking or pattern recognition could not be applied because of the fuzzy nature of images of FIM. A search for a suitable method led to correlation analysis. It was calibrated on Brownian motion and a known type of motion, such as cell marginal ruffling, compared with FIM. Results approved its explanatory potential. Therefore, several crucial incidences of FIM could be analyzed. Apart from an argument against viewing FIM as a manifestation of simple Brownian motion, the correlation analysis of FIM in the adjacent peripheries of a rat fibroblast and a K4 rat sarcoma cell confirmed the notion of higher and uneven distribution of velocity of FIM in a tumor cell so far shown in color-coded images only. This result and other yet unpublished observations indicate that the velocity and topology of FIM can also contribute to a biological distinction between neoplastic and normal cells. Regular application of the correlation analysis should further expand the study of FIM for its mechanisms and predictive value. Such an approach should be thoroughly examined for a contribution to the knowledge of cancer cells.