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Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice

Abstract

This protocol describes a methodology for imaging the sequestration of infected erythrocytes of the rodent malaria parasite Plasmodium berghei in the bodies of live mice or in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of infected erythrocytes is performed by measuring bioluminescence produced by the enzymatic reaction between luciferase and its substrate luciferin, which is injected into the mice several minutes prior to imaging. The bioluminescence signal is detected by an intensified charge-coupled device (I-CCD) photon-counting video camera. Sequestration of infected erythrocytes is imaged during short-term infections with synchronous parasite development or during ongoing infections. With this technology, sequestration patterns of the schizont stage can be quantitatively analyzed within 1–2 d after infection. Real-time in vivo imaging of infected erythrocytes will provide increased insights into the dynamics of sequestration and its role in pathology, and can be used to evaluate strategies that prevent sequestration.

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Figure 1: In vivo imaging system, IVIS100 from Xenogen, used for imaging luciferase-expressing sequestered schizonts in whole bodies of live mice or in isolated non-fixed organs.
Figure 2: Imaging of luciferase-expressing schizonts in a live mouse (a) Photographic image of a mouse obtained with the IVIS100 system.
Figure 3: Comparative analysis of bioluminescence intensities of two different mice.
Figure 4: In vivo imaging of sequestered schizonts in whole bodies of live mice with synchronized infections of the luciferase-expressing parasite line 354cl4.
Figure 5: Comparative analysis of bioluminescence signal intensities of isolated organs using the ROI option.

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Acknowledgements

We would like to thank H. Kroeze, J. Ramesar and S. Khan for their critical comments.

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Correspondence to Chris J Janse.

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Franke-Fayard, B., Waters, A. & Janse, C. Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice. Nat Protoc 1, 476–485 (2006). https://doi.org/10.1038/nprot.2006.69

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