A novel class of puromycin activity-based sensing probes containing analyte-specific responsive triggers have been synthesized and utilized for molecular imaging and histochemistry. After specific reaction between the trigger on the probe and target analyte, free puromycin molecules will be released and incorporated into nascent peptides. These incorporated puromycin can be detected after immunostaining, thus offering a highly sensitive method for detection of target analytes due to no leakage problem (as found in some reported fluorescent probes) and high signal-to-noise level from immunostaining. The syntheses of the probes are highly versatile, and representative examples for detection of reactive oxygen species (ROS), reactive sulfur species (RSS), reactive carbonyl species (RCS), ROS scavengers, and redox active metal ions have been demonstrated. One exemplary probe is Peroxymycin-1, which contains H2O2-responsive aryl boronate conjugated to puromycin through carbamate linkage. Peroxymycin-1 shows robust performance on molecular imaging of H2O2 in cell culture and histochemical analysis of H2O2 level in tissue samples harvested from small animals. It has been further employed for detection of elevated H2O2 level in liver tissues from a murine model of non-alcoholic fatty liver disease (NAFLD), suggesting its potential for studying disease pathology associated with H2O2 as well as disease diagnosis and monitoring of treatment progress.