The methods of the present invention exploit unique biochemical pathways present within infectious organisms to develop small molecule metabolic tracers. Labeled substrates created using these inventive methods were created. The labeled substrates can be used to determine whether a subject is infected with an infectious organism by imaging means, and with use of two or more such labeled substrates, methods of differentiating gram negative infection from gram positive infection, and methods of localizing and quantifying infectious disease burden are provided. The methods of the present invention can assist in the clinical decision to begin empiric antibiotic therapy, determine its efficacy, as well as the choice of antibacterial agents.