Fluorescence Lifetime Imaging as a Noninvasive Tool to Study Plasmodium Falciparum Metabolism.

ABSTRACT

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.