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Obtaining sufficient statistics in quantitative fluorescence microscopy is often hampered by the tedious and time-consuming task of manually locating comparable specimen and repeatedly launching the same acquisition protocol. Recent advances in combining fluorescence microscopy with online image analysis tackle this problem by fully integrating the task of identifying and locating the specimen of interest in an automated acquisition workflow. Here, we describe the general requirements and specific microscope control and image analysis software solutions for implementing such automated online feedback microscopy. We demonstrate the power of the method by two selected applications addressing high-throughput 3D imaging of sparsely parasite-infected tissue culture cells and automated fluorescence recovery after photobleaching experiments to quantify the turnover of vesicular coat proteins at ER exit sites.

Various murine lymphoid tumor cell lines were examined for their alterations in intracellular levels of cyclic AMP following exposure to several pharmacologic agents. The agents tested in the present study included 3-isobutyl-1-methylxanthine (MIX), isoproterenol (ISO), prostaglandin E2 (PGE), histamine, and cholera toxin (CT). B cell tumors were generally found to be less responsive to beta-adrenergic and PGE-induced increases in cyclic AMP than were T cell tumors. An exception to this general finding was the murine B lymphoma cell line WEHI-231, which demonstrated marked sensitivity to ISO and PGE. Macrophage tumors were generally found to be even less responsive to PGE than were the B cell tumors, again with some notable exceptions (P388.D1). Cholera toxin produced differential effects in B, T, and macrophage tumors, both in terms of the absolute magnitude of the cyclic AMP response and the kinetics of this response. A T lymphoma cell line (EL-4) was identified that seems to be totally unresponsive to cell surface receptor-mediated increases in intracellular levels of cyclic AMP. The results of this study are discussed in terms of the usefulness of murine lymphoid tumors for the study of pharmacologic modulation of the immune response by cyclic AMP-elevating agents. These results demonstrate the high degree of cellular heterogeneity that can exist within normal lymphoid populations of cells, and suggest that cloned cell lines may be useful in the biochemical characterization of subsets of lymphoid cells.