Optimization of malaria detection based on third harmonic generation imaging of hemozoin

Abstract

The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin.

Quantitative imaging of intraerythrocytic hemozoin by transient absorption microscopy

“…β-hematin was synthesized using an acid precipitation method documented previously. 26 Specifically, nitrogen gas was constantly pumped into 0.1 M sodium hydroxide for 15 min to deoxygenize the solution. Then 50 mg hemin chloride (Sigma Aldrich 51280) was dissolved in 10 ml deoxygenized sodium hydroxide solution and was kept away from light.…”

Third harmonic generation microscopy of cells and tissue organization

“…Transcutaneous THG detection of blood cell flow in dermal capillaries of human volunteers is sensitive enough to capture blood flow dynamics, and the ratios of erythrocytes and white blood cells ; this can be exploited for in vivo cytometry and hematological analysis without the need to draw blood. In malaria-infected blood cells, THG is particularly enhanced owing to the altered scatter properties that are caused by hemozoin, a parasite-derived pigment (Tripathy et al, 2013); this facilitates sensitive and rapid ex vivo detection of early stage infection in blood cells and thus has the potential to replace currently used skill-and time-intensive molecular analysis (Bélisle et al, 2008). The above studies clearly demonstrate the promise of THG to be used to detect interface changes at the microscale in the clinical diagnosis of various conditions.…”

Harmonic Generation Microscopy 2.0: New Tricks Empowering Intravital Imaging for Neuroscience

“…Since an initial demonstration of THG enhancement by the absorption band of hemoglobin (Clay et al, 2006), packaged porphyrins have been proved as a THG agent (Cui et al, 2015). Also, the hemozoin pigment, a natural crystalline aggregate of hemes produced by parasites, emits a strong THG signal which can be exploited for detecting malaria (Belisle et al, 2008; Tripathy et al, 2013). However, given its famous role as a generator of singlet oxygen, porphyrin-based THG imaging raises a concern of photosafety.…”