![]() This subsequently poses a trade-off between temporal resolution and maximum measurement time as imaging rates and setups are usually chosen at the beginning of the experiment. A more severe problem is posed by phototoxicity ( 2) and probe photobleaching ( 21), leaving the researcher with a limited sampling rate. The long lag period produces a high and error-prone workload for the researcher as large image stacks have to be processed and stored. Specifically, these studies may last up to several days because key signaling events often happen unpredictably or after a long lag period, but can then proceed rapidly once initiated ( 19, 20). However, single-cell studies such as cell proliferation ( 17, 18) and studies of programmed cell death (apoptosis) pose a number of challenges to live cell imaging. Applications of the method range from drug toxicity studies ( 13) to investigations of biochemical signal transduction and its application to systems biology studies ( 14–16). Employing fluorescent protein variants with different excitation/emission wavelengths, Förster (or fluorescence) resonance energy transfer (FRET) can be utilized to study protein-protein interactions, protein-DNA interactions, and protein conformational changes ( 11, 12) within a living cell. Likewise, cloned DNA can be introduced into cells to fluorescently label intracellular proteins of interest by marker proteins such as GFP, its spectral variants, and related fluorescent proteins ( 6, 10). Today, synthetic fluorescent sensors can be applied to measure a number of different physiological parameters such as plasma and mitochondrial transmem-brane potentials ( 4, 5), ion concentrations ( 6, 7), or intracellular pH ( 8, 9). With the improvements in imaging technology and the ever-increasing number of novel probes, confocal time-lapse imaging has become a powerful tool for studies of living cells in biomedicine and systems biology ( 1–3). The demonstration provides a clear proof-of-concept for ALISSA, and offers guidelines for its application in a broad spectrum of signal transduction studies. We have applied the ALISSA framework to the analysis of apoptosis as a demonstration case for slow onset and rapid execution signaling. It consists of a reusable image analysis software for cell segmentation, tracking, and time series extraction, and a measurement-specific process control software that can be easily adapted to various biological settings. The system employs online image analysis to detect cellular events that are then used to exercise microscope control. It allows an adaptation of image modalities and laser resources tailored to the biological process, and thereby extends temporal resolution from minutes to seconds. We have developed ALISSA, a design framework and reference implementation for an automated live-cell imaging system for signal transduction analysis. Consequently, when a study of cellular processes requires measurements over hours or days, temporal resolution is limited, and spontaneous or rapid events may be missed, thus limiting conclusions about transduction events. Probe photobleaching and a specimen's sensitivity to phototoxicity severely limit the number of possible excitation cycles in time-lapse fluorescent microscopy experiments. ![]()
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