Sensitive and label-free malaria detection

David L. Shenkenberg

Malaria diagnosis currently involves either antibody-based rapid diagnostic tests or microscopic analysis using the classical Giemsa stain, but there are drawbacks to using these methods in the indigent, tropical countries where the disease remains a serious problem. Rapid diagnostic tests use antibodies that tend to degrade in the tropical heat and that may not detect all strains of the malaria parasite, whereas Giemsa microscopy requires a labeling step and time-consuming analysis performed by a trained microscopist. In contrast, detection using third harmonic generation (THG) does not require reagents or a labeling step, and it likely will not require a highly trained operator.

Principal investigator Paul W. Wiseman and colleagues from McGill University in Montréal have investigated the efficacy of malaria detection using third-harmonic-generation detection of hemozoin, a substance produced by the Plasmodium protozoan blood parasite that causes malaria. In the process of feeding on the oxygen-carrying hemoglobin protein present in red blood cells, the parasite converts the toxic heme group of hemoglobin into hemozoin crystals that become stored in its food vacuole. Three photons from the excitation source that simultaneously hit the hemozoin convert into one photon with triple the frequency, or the third harmonic of the incident excitation light.

Researchers imaged red blood cells infected with the malaria parasite Plasmodium falciparum. The right panels show cells imaged using bright-field microscopy with classical Giemsa staining, and the left panels show pseudocolor third harmonic imagesof hemozoin (blue) superimposed on two-photon autofluorescence from red blood cells. Hemozoin is produced by Plasmodium as it digests red blood cells. Images reprinted with permission of Biophysical Journal.

Wiseman suspected that these hemozoin crystals might exhibit a third harmonic response because he had previously imaged heme and chlorophyll, substances that can exhibit strong third harmonic emission and that share ring-shaped chemical structures known as porphyrins. Hemozoin crystals have the same porphyrin rings because they are the heme packaged by the parasites. A paper that details the researchers’ experiments with hemozoin appears in the Dec. 7 online edition of Biophysical Journal.

Hemozoin third harmonic

The researchers detected the hemozoin third harmonic in red blood cell samples infected with either of two strains of Plasmodium falciparum, using an imaging system assembled by graduate student Jonathan M. Bélisle. As with other multiphoton excitation phenomena, THG requires a setup that focuses high-intensity pulsed laser light so that the photons are densely crowded in both space and time.

The investigators induced the third harmonic with a Coherent optical parametric oscillator, which has a usefully high repetition rate for rapid imaging. It was pumped by a femtosecond Ti:sapphire laser. This also induced two-photon autofluorescence from the red blood cells, a side benefit that allowed the localization of the THG emission within autofluorescence images of the cells. They used a beamsplitter to separate the third harmonic and the two-photon autofluorescence signals, and each traveled to a photomultiplier tube. The scientists mapped the detected signals point by point to construct pseudocolor images.

The researchers used a self-assembled setup consisting of an optical parametric oscillator (OPO) pumped by a femtosecond Ti:sapphire laser to generate the third harmonic from hemozoin and two-photon autofluorescence from red blood cells. These signals were separated by a beamsplitter, and each traveled to a photomultiplier tube (PMT).

Third harmonic signals typically are much less intense than fluorescence signals, yet when Bélisle imaged the hemozoin, he found that the signal was so strong it almost damaged the photomultiplier tubes and that the emission had to be attenuated. Wiseman said that it is the most intense signal the researchers have observed using third harmonic generation. They achieved signal-to-noise ratios as great as 1000:1 for the THG from the hemozoin in infected blood cells with late-stage parasites.

In an earlier stage of the parasite life cycle known as the ring stage, they found that the hemozoin exhibited a lower signal-to-noise ratio, albeit still one comparable to those achievable with fluorescence detection. The ratio ranged from 30:1 to 100:1, with a mean of 62:1. Wiseman said that the lower ratio makes sense because more of the porphyrin-containing heme becomes incorporated into the hemozoin structure as the parasite develops and digests more hemoglobin.

When the investigators compared their results with other empirical results and to a theoretical paper on THG authored by members of Xiaoling Sunney Xie’s laboratory at Harvard University in Cambridge, Mass., they found that the hemozoin crystals have ideal properties for THG detection; hence, the high signal-to-noise ratios. As in the theoretically ideal case, the size of hemozoin crystals is close to the excitation wavelength, and the porphyrin rings in hemozoin have polarizable pi electron bonds.

The researchers believe that a THG-based detection system incorporated into a flow cytometer would enable label-free malaria detection. They speculate that an operator simply could inject blood samples into the flow cytometer and receive a reading of the total cell count and the number of infected cells. Wiseman said that this would minimize overdiagnosis and treatment with harsh malaria drugs, as anecdotal evidence suggests that health practitioners in endemic countries sometimes bypass the diagnosis step and broadly treat patients as if they are infected. It also could reduce the cost of diagnosis for clinical applications if an excitation source could be produced at a reasonable cost.

He said that, for a more flexible instrument, it also might be possible to integrate a THG system into a fluorescence flow cytometer because fluorescence often is collected in the back direction and the third harmonic in the forward direction.

The researchers plan to look at more strains of malaria throughout the entire parasite life cycle, and they currently are investigating the dependence of the third harmonic on the size of synthetically produced hemozoin crystals.