Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.
Keywords: Bioluminescence; Chronic Chagas disease; Confocal microscopy; Fluorescence; In vivo imaging; Murine models; Trypanosoma cruzi.