Summary Malaria continues to be a global health priority, with almost half of the world?s population (3.2 billion) at risk and around 500,000 deaths each year. Overtreatment is caused by lack of diagnosis and contributes to resistance, a huge threat as illustrated by artemisinin resistance in Southeast Asia. Little progress has been made in the novel methods to diagnose malaria, let alone in reliably identifying asymptomatically infected individuals, which is of utmost importance for the current elimination agenda. Most current malaria tests have poor sensitivity at low levels of parasitemia: in routine practice it is usually well above 0.001% or ? 50 parasites/L. That high a level is insufficient to detect the large reservoir of asymptomatic individuals. The goal of this proposal is to develop an innovative multicolor malaria detector using malaria-specific in vivo photoacoustic (PA) flow cytometry (PAFC) platform for the early, noninvasive (needle-free, no blood draw and thus no contamination concern), label-free (no injection of any reagent into the blood), rapid (a few seconds), and safe (no pain or skin damage) diagnosis of malaria. This technology is based on PA detection of the malaria crystal (hemozoin) within circulating infected red blood cells (RBCs) directly in the bloodstream. Transcutaneous irradiation of hand vessels with a low pulse-energy laser leads to the microscopic local heating of hemozoin accompanied by thermoacoustic generation of acoustic waves, recorded with an ultrasound transducer attached to the skin. The high acoustic resolution and high-speed multicolor PAFC can detect and identity hemozoin in deep, large, high flow-rate blood vessels to provide the earliest and fastest malaria diagnosis within 20 seconds at an unprecedented parasitemia threshold as low as 0.0000001% (5 iRBCs/mL). This is 1,000-10,000 times better than existing clinical malaria tests. This goal will be achieved through two interconnected specific aims: (1) Develop new malaria-specific PAFC diagnostic device prototype; (2) Determine the efficacy of PAFC devices for label-free detection of iRBCs in a preclinical mouse model. The PAFC device will not be affected by hand movement or skin pigmentation. This malaria-specific PAFC technology can be used to identify asymptomatic malaria-infected individuals in a short time through gentle contact of the PA probe with the intact skin. The PAFC device can be a unique tool for malaria elimination by identifying asymptomatically infected humans, which allows targeted interventions with appropriate treatment. Overall, this method will contribute substantially to allow treatment only for those who truly are infected, while avoiding unnecessary and inappropriate overtreatment of other febrile illnesses.