Subcellular Fluorescence in Drug Response Studies: Interference Controls and Limits of Biological Inference

Subcellular fluorescent reporters are used to select compounds and investigate drug mechanisms, but treatment can change reporter delivery, chemistry and biological effects as well as the intended measurand. This critical narrative review examines how those alternatives affect decisions in intracellular biosensing assays. We synthesize representative methodological and pharmacological studies across six cellular compartments and compare the scope of existing reviews of probe design, drug visualization and enzyme-specific validation. Published examples connect oxidation-product identification, calibration-model dependence and state-dependent localization to the conclusions each measurement can support. A decision matrix links competing explanations to informative countertests and specifies when a result supports only an optical phenotype, a compartmental measurement or a functional response. Drug-by-probe-by-illumination comparisons and independent functional endpoints are used to separate apparent optical rescue from biological recovery. Screening applications require an additional test of whether reporter features improve prediction beyond cell-count or viability baselines. The proposed workflow integrates established controls for a defined pharmacological decision; it is not a new validated method or a reporter ranking. Its practical purpose is to make the interpretation of discordant measurements explicit and to identify which additional experiment could change compound prioritization.