Probe photobleaching and a specimen's sensitivity to phototoxicity severely limit the number of possible excitation cycles in time-lapse fluorescent microscopy experiments. 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. We have developed ALISSA, a design framework and reference implementation for an automated live-cell imaging system for signal transduction analysis. It allows an adaptation of image modalities and laser resources tailored to the biological process, and thereby extends temporal resolution from minutes to seconds. The system employs online image analysis to detect cellular events that are then used to exercise microscope control. 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. We have applied the ALISSA framework to the analysis of apoptosis as a demonstration case for slow onset and rapid execution signaling. The demonstration provides a clear proof-of-concept for ALISSA, and offers guidelines for its application in a broad spectrum of signal transduction studies.

Introduction

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 (1,2,3). Today, synthetic fluorescent sensors can be applied to measure a number of different physiological parameters such as plasma and mitochondrial transmembrane potentials (4,5), ion concentrations (6,7), or intracellular pH (8,9). 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). 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. Applications of the method range from drug toxicity studies (13) to investigations of biochemical signal transduction and its application to systems biology studies (14,15,16). 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. 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). The long lag period produces a high and error-prone workload for the researcher as large image stacks have to be processed and stored. A more severe problem is posed by phototoxicity (2) and probe photobleaching (21), leaving the researcher with a limited sampling rate. 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. This dilemma is aggravated when the desired events are themselves rapid, but occur unpredictably and late after stimulus onset. Premature phototoxicity/photobleaching then limits the sampling rate and biological events may be entirely overlooked or resolved only at a temporal resolution that is too low. While there have been significant efforts to reduce these drawbacks by probe optimization (22,23), we present here a method that allows the economic use of laser excitation and imaging resources in a manner that is tailored to the stage of the experiment when they are required. Our approach employs cell segmentation, cell tracking, and time series evaluation online and in real time (e.g., during experimentation) to detect temporal onset of cellular events and change microscope image modalities subsequently. We present a software framework that separates process control from image analysis, rendering the latter reusable for a broad spectrum of applications. Proof-of-concept is provided by applying the framework to signal transduction studies during apoptosis.

Materials and methods

Time-lapse microscopy and digital imaging

For the ALISSA proof-of-concept demonstration, cells were cultured in glass-bottom dishes (Cat. no. KIT-3512; Willco B.V., Amsterdam, The Netherlands) and equilibrated with 30 nM tetramethyl-rhodamine methyl ester (TMRM) in 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid–buffered (10 mM; pH 7.4) RPMI (both, Sigma-Aldrich, Arklow, Ireland) containing 10% FCS, covered with mineral oil, and placed in heated (37°C) incubation chambers (Tempcontrol Digital with Objective Heater and Heating Insert P; Carl Zeiss, Jena, Germany) that were mounted on the microscope stage. Apoptosis was induced with 1 µM staurosporine (STS) (Cat. no. ALX-380-014-M001; Alexis Corporation, Exeter, UK), a broad-spectrum kinase inhibitor. The confocal microscope used was a Zeiss LSM 510 META inverted microscope (Carl Zeiss) equipped with a 405-nm GaN laser, 488-nm argon laser, and a 543-nm helium/ neon laser. Using a 63 × 1.4 numerical aperture (N.A.) oil immersion objective, cyan fluorescent protein (CFP), FRET, yellow fluorescent protein (YFP), and TMRM fluorescence were measured using the appropriate filters and beam splitters. Acquisition channels and wavelengths are depicted in Table 1. Mitochondrial membrane potential depolarization accompanied by mitochondrial outer membrane permeabilization (MOMP) was detected by delocalization of the cationic fluorescent dye TMRM and using image analysis techniques as described in the “Time series extraction (IAS)” section. Cells were transfected with pCFP-DEVD-YFP construct to visualize effector caspase activity by FRET (24). Images were stored separately for each acquisition setting as 8-bit grayscale in TIFF format.