Microscopy environment for quantitative spatial and temporal analysis of multicellular interactions

Quantitative analysis of spatial and temporal concurrent responses of multiple markers in 3-dimensional cell cultures is hampered by the routine mode of sequential image acquisition, measurement and analysis of specific targets. A system was developed for detailed analysis of multi-dimensional, time-sequence responses and in order to relate features in novel and meaningful ways that will further our understanding of basic biology. Optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating as described by Lanni. The automated microscopy system can image multicellular structures and track dynamic events, and is equipped for simultaneous/ sequential imaging of multiple fluorescent markers. Computer-controlled perfusion of external stimuli into the culture system allows (i) real-time observations of multiple cellular responses and (ii) automatic and intelligent adjustment of experimental parameters. This creates a feedback loop in real-time that directs desired responses in a given experiment. On-line image analysis routines provide cell-by-cell measurement results through segmentation and feature extraction (i.e. intensity, localization, etc.), and quantitation of meta-features such as dynamic responses of cells or correlations between different cells. Off-line image and data analysis is used to derive models of the processes involved, which will deepen the understanding of the basic biology.