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Abstract Being able to quantify the phototoxicity of dyes and drugs in live cells allows biologists to better understand cell responses to exogenous stimuli during imaging. This capability further helps to design fluorescent labels with lower phototoxicity and drugs with better efficacy. Conventional ways to evaluate cellular phototoxicity rely on late‐stage measurements of individual or different populations of cells. Here, we developed a quantitative method using intracellular microtubule polymerization as a rapid and sensitive marker to quantify early‐stage phototoxicity. Implementing this method, we assessed the photosensitization induced by organelle dyes illuminated with different excitation wavelengths. Notably, fluorescent markers targeting mitochondria, nuclei, and endoplasmic reticulum exhibited diverse levels of phototoxicity. Furthermore, leveraging a real‐time precision opto‐control technology allowed us to evaluate the synergistic effect of light and dyes on specific organelles. Studies in hypoxia revealed enhanced phototoxicity of Mito‐Tracker Red CMXRos that is not correlated with the generation of reactive oxygen species but a different deleterious pathway in low oxygen conditions.

Abstract Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, a closed‐loop optoelectronic control system is developed that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical‐selective fluorescence signals are utilized to command lasers that trigger specific chemical processes or control the activation of photoswitchable inhibitors at desired targets. This technology is fully compatible with laser scanning confocal fluorescence microscopes. The authors demonstrate selective interactions of a 405 nm laser with targeted organelles and simultaneous monitoring of cell responses by fluorescent protein signals. Notably, blue laser interaction with the endoplasmic reticulum leads to a more pronounced reduction in cytosolic green fluorescent protein signals in comparison to that with nuclei and lipid droplets. Moreover, when combined with a photoswitchable inhibitor, microtubule polymerization is selectively inhibited within the subcellular compartments. This technology enables subcellular spatiotemporal optical manipulation over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non‐targeted locations.

Chemical imaging via advanced optical microscopy technologies has revealed remarkable details of biomolecules in living specimens. However, the ways to control chemical processes in biological samples remain preliminary. The lack of appropriate methods to spatially regulate chemical reactions in live cells in real-time prevents investigation of site-specific molecular behaviors and biological functions. Chemical- and site-specific control of biomolecules requires the detection of chemicals with high specificity and spatially precise modulation of chemical reactions. Laser-scanning optical microscopes offer great platforms for high-speed chemical detection. A closed-loop feedback control system, when paired with a laser scanning microscope, allows real-time precision opto-control (RPOC) of chemical processes for dynamic molecular targets in live cells. In this perspective, we briefly review recent advancements in chemical imaging based on laser scanning microscopy, summarize methods developed for precise optical manipulation, and highlight a recently developed RPOC technology. Furthermore, we discuss future directions of precision opto-control of biomolecules.