4-DIMENSIONAL IMAGING OF LIVING CHONDROCYTES IN CARTILAGE USING CONFOCAL MICROSCOPY - A PRAGMATIC APPROACH

Regulation of cell volume is a fundamental cellular homeostatic mechan ism in the face of osmotic stress. In normal articular cartilage, chon drocytes are exposed to a changing osmotic environment. We present a c omprehensive protocol for studying the volume regulatory behavior of c hondrocytes within intact cartilage tissue using confocal laser-scanni ng microscopy. Our data acquisition regime optimizes both signal-to-no ise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated co mponent of the imaging system, and we demonstrate methods for the dire ct assessment of tissue-induced axial attenuation and image distortion . Parameterized functions describing these two components of image deg radation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualizatio n of four-dimensional (4-D) images. We have devised two new types of d ata reconstruction. The first compresses each 3-D time point into a si ngle quantitative view, termed a coordinate view From these reconstruc tions we are able to simultaneously view and extract cell measurements . A second type, a 4-D reconstruction, uses color to represent relativ e changes in cell volume, again while maintaining the morphological an d spatial information. Both these approaches of image analysis and vis ualization have been implemented to study the morphology, spatial dist ribution, and dynamic volume behavior of chondrocytes after osmotic pe rturbation. We have mapped chondrocyte shape, arrangement, and absolut e volume in situ, which vary significantly from the tissue surface thr ough to the underlying bone. Despite the rigid nature of the extracell ular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques o f confocal laser-scanning microscopy and vital cell labeling have enab led us to study, for the first time, the response of chondrocytes in s itu to changes in interstitial osmotic pressure.