Chapter 2 │ Page 37 1. INTRODUCTION Live-cell imaging with fluorescence microscopy is a powerful and convenient method for real-time, high-throughput capturing of dynamic cellular processes and treatment responses. This type of imaging uses in situ time-lapse microscopy to provide spatiotemporal visualization and quantification of living biological samples via light excitation of specific fluorophores [1]. By removing the need for fixation or dissociation of the cell culture, fluorescence live-cell imaging has advantages over other standard read-out and detection methods, including high sensitivity and selectivity, improved resolution and reproducibility, and automation and high-throughput capacities [2]. Furthermore, a wide range of fluorescence labelling techniques (e.g. chemical, enzymatic, protein/peptide tagging) are currently available, enabling the tagging of practically all subcellular compartments and structures [3, 4]. Based on application goals and biological targets, researchers can choose between transient or stable labelling of the sample. While transient labelling is ideal for rapid read-outs of monocultures, permanent incorporation of a reporter (e.g. via viral transduction or transfection), allows for the establishment of a stable fluorescent clone, thus enabling long-term analysis, screening assays, and cell tracking in relevant co-culture models [5, 6]. These appealing properties have facilitated the adoption of this versatile analytical tool in various research fields, including developmental biology, neurology, and oncology [7]. Nevertheless, a major drawback of this technique is the potential for light-induced toxicity, known as phototoxicity, which has often been drastically underestimated [8-12]. Cell cultures maintained in vitro are typically not adapted to coping with repetitive excitation of incorporated fluorophores by common sources of light, thus resulting in additional cellular stress, damage, or even cell death [9, 11]. Disruption of homeostasis by phototoxicity can be a major confounding factor in test samples,
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