The outward manifestation of our physiology in health or disease is the culmination of an array of sub-cellular events driven by molecular machines. These machines range from large enzymes to reactive small molecules, all of which exert an effect on the cell to alter its function for better or for worse. These active biomolecules rarely work in isolation, but are rather parts of integral networks within which any single sub-cellular machine is only transiently active or transiently present, giving way to its successor in the signaling chain. This transiency often renders these biomolecular machines elusive to analysis in the context of the living organism. Measuring the activity of these molecules and overcoming the difficulty of target transiency can enable both the detection of disease prior to outward signs and symptoms, and the assessment of the success or failure of therapy prior to disease progression. Molecular imaging is a powerful tool for assessing a target biomolecular machine non-invasively and longitudinally over an entire volume of interest in living subjects. New chemistries for activatable molecular imaging probes and the implementation of these probes in living animals will be described, focusing on the detection of three transient biomolecular machines turned on early in subcellular signaling cascades. Reactive oxygen species (ROS) are highly reactive oxygen-centered biomolecules underlying a broad range of diseases. In the context of injury, ROS are very early effectors of the tissue response to the source of harm. Novel optical nanoprobes have been developed from semiconducting polymers that can sensitively report on the presence of specific ROS in living mice [1,2]. The application of these nanoprobes to the real-time detection of the very early response to sterile tissue injury, as well as the real-time assay of drug-induced hepatotoxicity in living animals will be discussed. This nanoprobe enables ROS assays in vivo that were previously confined to the in vitro space. Poly (ADP ribose) polymerase-1/2 (PARP-1/2) is an enzyme that monitors DNA integrity, where PARP-1/2 activity acts as an intracellular signal selecting between DNA repair or cell death. PARP enzymes catalyze the polymerization of nicotinamide adenine dinucleotide (NAD), a transient polymer product that can be degraded within minutes of its formation by specific glycohydrolases. The development of a substrate-based radiotracer for the mapping of PARP-1/2 activity by positron emission tomography (PET) will be discussed, as well as its implementation in animal models of radiation therapy against breast or cervical cancer [3]. PARP-1/2 activation was detected hours after low dose radiation therapy, as was enzyme inhibition following PARP inhibitor therapy. This unprecedented in vivo investigation of PARP-1/2 revealed differences in the timing of therapy response between tumor types, suggesting that timing of interventions are tumor type-specific. Caspase-3 is a cysteine-aspartate protease whose activation signals the committal of the cell to die through apoptosis, a common death pathway induced by a variety of cancer chemotherapeutics and radiation. A modular probe was designed to undergo bioorthogonal macrocyclization upon activation by caspase-3, with the macrocycles self-assembling into nanoparticles in situ in tumor tissue [4,5,6,7]. Since caspase-3 activity correlated with the degree of probe retention in dying tumor tissue, this molecular imaging strategy was applicable to optical, PET, and magnetic resonance imaging (MRI). This self-assembling molecular imaging probe was able to detect apoptosis induced by doxorubicin or radiation therapy in tumor-bearing mice following systemic administration of contrast agent. Enhanced contrast production following therapy was not detected using a non-activatable control analog, illustrating the utility of our bioorthogonal self-assembly probe design for therapy response monitoring. In discussing these three transient biomolecular machines as targets of molecular imaging, a variety of probe chemistries will be introduced and the ability to obtain non-invasive, longitudinal, and predictive data about sub-cellular physiology within the intact organism will be demonstrated. The molecular imaging of these key sub-cellular machines is sure to continue to provide an unprecedented level of interrogation of physiology to both enhance our investigations of health and disease, and improve clinical outcomes.