Evolution and efficiency of cell death detection methods: From classical assays to intelligent systems

Detecting cell death is a fundamental pillar of contemporary biomedical studies, which supports the diagnostics of a disease, its therapeutic assessment, toxicological tests, and mechanistic biology. The cell death methodologies have evolved over the last seven decades, being based on descriptive morphological evaluations up to a highly quantitative, pathway-specific, and automated methodology. The pioneering microscopic methods offered had only initial information as they lacked sensitivity and objectivity. Molecular confirmation of apoptosis was subsequently achieved by subsequent biochemical assays DNA fragmentation and TUNEL, although at the later stages. With the introduction of flow cytometry there was the introduction of multiparametric and high throughput detection which greatly enhanced efficiency in terms of accuracy and scalability. Simultaneously, the molecular marker-based assays had a mechanistic specificity, although they added complexity to the experiment. Most recently, label-free imaging, microfluidic systems and artificial intelligence-based systems have proven to be potent tools that can detect various modalities of cell death, including apoptosis, necroptosis, pyroptosis, and ferroptosis, in real-time, non-invasively and predictively. The review systematically chronologically tracks the development of cell death detection technologies, critically assesses their performance in terms of sensitivity, specificity, and ability to perform a temporal read out, and points out new innovative technologies that are likely to revolutionize cell death analysis in translational and clinical researches.