QUANTITATION OF CELL-MATRIX ADHESION USING CONFOCAL IMAGE-ANALYSIS OFFOCAL CONTACT ASSOCIATED PROTEINS AND INTERFERENCE REFLECTION MICROSCOPY

We have developed an approach for the quantitation of vinculin, a foca l contact associated protein, based on a multimodal confocal microscop y and image analysis. Vinculin spot distribution mas imaged in confoca l fluorescence microscopy and the corresponding focal contacts were im aged in confocal interference reflection microscopy. These images were analyzed with a SAMBA image cytometer. The image analysis program pro vided 12 morphometric features describing cellular area, shape. and pr oportions of vinculin spots as well as six topographical features desc ribing the distribution of vinculin and the relative overlap of vincul in and focal contacts. This approach was applied to the study of rat o steosarcoma cells submitted to mechanical stresses: successions of 2g and Og accelerations during a series of parabolic flights. The measure d features were assessed by means of correlation analysis and stepwise discriminant analysis. After correlation analysis, only ten parameter s were retained. Quantitation of cell morphological parameters indicat ed that cell area was significantly affected by gravitational stresses as well as vinculin distribution. Cell area was reduced by 50% and vi nculin spots were restricted to cell periphery. Cell adhesion measured by IRM decreased significantly in the first part of the flight nod re mained stable at the end of the flight. These results suggest that cel l-matrix adhesion is affected by gravitational stresses. image analysi s provides useful tools to investigate focal adhesion re-organization under different physiological stimuli. (C) 1997 Wiley-Liss, Inc.