Real-time single cell analysis is necessary to understand dynamic cellular functions in time and space. Such analyses require the simultaneous measurement of multiple variables in real-time, due to heterogeneity in cellular populations. We report the application of using a micro-environmental chamber on an automatic laser scanning confocal microscope to observe murine macrophage cells in incubation conditions for more than 18 hours. The motorized stage of the microscope was programmed to scan through pre-defined monitoring locations to increase the observation throughput. The acquired images were post-processed to extract the information of each cell. In contrast to current single-cell technologies, such as fluorescence-activated cell sorter (FACS) based systems, the reported architecture records the history of the physiological responses of individual cells.