Anal Bioanal Chem (2013) 405:5431–5440 DOI 10.1007/s00216-013-6985-z
RESEARCH PAPER
Optimization of malaria detection based on third harmonic generation imaging of hemozoin Umakanta Tripathy & Maxime Giguère-Bisson & Mohammad Hussain Sangji & Marie-Josée Bellemare & D. Scott Bohle & Elias Georges & Paul W. Wiseman
Received: 4 January 2013 / Revised: 10 April 2013 / Accepted: 12 April 2013 / Published online: 7 May 2013 # Springer-Verlag Berlin Heidelberg 2013
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. Keywords Biomaterials . Nonlinear microscopy . Third harmonic generation . Imaging . Malaria . Hemozoin . Polarization U. Tripathy : M. H. Sangji : P. W. Wiseman Department of Physics, McGill University, 3600 University St., Montreal, QC H3A 2T8, Canada M. Giguère-Bisson : M.-J. Bellemare : D. S. Bohle : P. W. Wiseman (*) Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, QC H3A 2K6, Canada e-mail:
[email protected] E. Georges Institute of Parasitology, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Montreal, QC H9X 3V9, Canada
Introduction Malaria is a major disease in tropical countries affecting one third of the world’s population [1]. During the blood stage of the life cycle, the malaria parasites digest hemoglobin in the host red blood cells that leads to the release of large quantities of free heme. The heme is toxic to the parasite if not crystallized into insoluble hemozoin crystals inside the food vacuole. The deposited hemozoin, also known as the malaria pigment, is eventually released from ruptured red blood cells and can remain undegraded within the host for long periods of time [2]. The detoxification of heme, which has a pleotropic effect on lipid membranes [3] via formation of hemozoin crystals, is essential for the survival of the malaria parasites inside the host red blood cells, and consequently, it is an attractive target for both the detection of the parasites and the development of antimalarial drugs. Several techniques have been used for the detection of malaria in blood samples including: Giemsa staining of the parasite DNA followed by examination of the samples with light microscopy, fluorescent DNA/RNA stains, malaria pigmentation detection, molecular methods, and antigen testing [4–10]. In addition, recently, we have demonstrated that third harmonic generation (THG) imaging is highly sensitive and specific for the detection of malaria infection due to the strong nonlinear optical response of the hemozoin crystals within parasites inside the infected host red blood cells [11]. In our earlier report [11], the signal-to-noise (S/N) ratio was measured to be as high as 1,000:1 for the crystals in the trophozoite stage, and in the ring stage, it varied from 30 to 100 with a mean of 62. However, the complete parameter space for optimization of THG response from hemozoin was not systematically explored. THG is a parametric nonlinear process that involves the conversion of three incident photons into one emitted photon of three times the fundamental laser frequency due to the
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third-order dielectric susceptibility (χ(3)) of the medium. The virtual energy conservation nature of THG (in contrast to fluorescence) has advantages for live cell and clinical microscopy applications. THG is purely a materialdependent optical effect and provides inherent optical sectioning due to the nonlinear response which restricts its production inside the tight focus of a femtosecond pulsed laser beam. In addition, the tightly focused femtosecond laser beam slightly modifies the molecular orientation that leads to the generation of phase-matched THG from the focal point. Here, the phase-matching condition is required for the generation of a strong THG signal for materials larger than the excitation wavelength and it is more easily met in birefringent anisotropic materials either by angle tuning or by temperature tuning. Also, no saturation or photo-bleaching in the generated signal is expected due to the nature of THG when imaging off-resonance. THG laser scanning microscopy has been applied to study a number of materials and biological samples including transparent objects [12], gold nanoparticles [13], hemozoin [11], Chara plant rhizoids [14], Clivia miniata leaf chloroplasts and flowing erythrocytes [15], mitochondria in rat cardiomyocytes [16], lipid bodies in cells and tissues [17], and myelin in the central nervous system [18]. In this study, we systematically explore a broader parameter space for THG nonlinear microscopy imaging of natural hemozoin crystals found in human red blood cell samples infected with FCR-3 Plasmodium falciparum trophozoites as well as synthesized isostructural crystals of hematin anhydride with dimensions ranging from ∼0.37 to 3.98 μm. We explore the range of THG response from the hemozoin crystals for variable excitation laser wavelength, incident laser power, as well as the different excitation laser polarization states. This characterization of the parameter space for imaging of THG signal from hemozoin crystals is an essential first step for optimization of malaria detection strategies using this modality.
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than 150 fs pulses at a laser pulse repetition rate of 76 MHz. In this study, we imaged THG signal from hemozoin crystals for excitation wavelengths ranging from 1,140 to 1,260 nm and found optimal response at 1,170 nm excitation from the OPO corresponding to a pulse width of ∼97 fs. Laser beam scanning is achieved with a VM2000 moving magnet scanner (GSI Lumonics, Moorpark, CA, USA). The excitation and collection objectives (Carl Zeiss, Toronto, Canada) are a 63×, 0.9 numeric aperture (NA) water immersion lens with a 2-mm working distance and a 20×, 0.8 NA water immersion lens with a 0.61-mm working distance, respectively. The sample holder can be manually moved in X–Y and is controlled in the Z direction by a piezoelectric actuator (Thorlabs, Newton, NJ, USA). For light detection, two H7422P-40 photomultiplier tubes (Hamamatsu Photonics, Bridgewater, NJ, USA) were used for simultaneous detection of THG and two-photon (TP) fluorescence signal in two channels with splitting via a dichroic mirror (Chroma, 540dcxr that transmits TP signal and reflects THG signal). Two cutoff filters were used to select the signal detection range for the THG (Chroma, HQ400/40m-2p) and TP (Chroma, HQ600/50m-2p). The analog-to-digital conversion of the signal from the PMT is done using a 16-bit DAQ (National Instruments, Austin, TX, USA) which can acquire 1.25 Msamples/s. Instrument control and image acquisition were done with custom written LabVIEW programs. Full details on the multimodal nonlinear laser scanning microscope were described previously [19]. The excitation laser polarization experiments were carried out by inserting a half wave Fresnel Rhomb Retarder (FR600HM, Thorlabs, USA) in the laser path. We also systematically studied the THG response from the hemozoin samples as a function of laser power as controlled by neutral density filters with different transmission percentages. Sample preparation Malaria-infected red blood cell preparation
Methods and materials Multimodal nonlinear laser scanning microscopy The images were recorded using a custom-built multimodal nonlinear laser scanning microscope. The laser source for this microscope is a Mira optical parametric oscillator (OPO) (Coherent, Santa Clara, CA, USA) that is linearly polarized in the horizontal direction. The OPO is pumped at a 780-nm wavelength by a Mira 900 Ti:Sapphire (Coherent) at an average power of 1.9 W, which itself is pumped by a V18 Verdi laser (Coherent) at 13 W average power. The emission wavelength of the OPO is tunable from 1,000 to 1,300 nm at an average power of 300 mW, which gives less
P. falciparum strain (FCR-3) was grown in vitro on washed human erythrocytes (type B+) from freshly drawn blood (suspended at 5 % hematocrit) in culture medium (RPMI1640 from Gibco supplemented with 0.5 % Albumax II, 0.32 mM hypoxanthine, 2 mM L -glutamine, 25 mM HEPES, 24 mM sodium bicarbonate, 11 mM glucose). Parasite culture (10 mL in T-25 tissue culture flask) was flushed with 5 % CO2 and incubated at 37 °C with constant shaking and later synchronized with 5 % D-sorbitol for 7 min followed by 3 min centrifugation at 1,800 rpm. Synchronized parasite pellets were resuspended in fresh RPMI tissue culture media with 10 % serum. Samples of infected red blood cells were chemically fixed before being sealed in between two glass coverslips for imaging.
Optimization of malaria detection based on third harmonic generation
Hematin anhydride synthesis To perform the crystal size studies, the synthesis of the crystals was varied to generate different sizes (length, width, and thickness) by the following protocols. In general, either the acid precipitation or anhydrous base-catalyzed preparations were used [20], and a variation using acetone as the solvent is outlined below.
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characterized by UV–visible absorption, X-ray diffraction, infrared, and field emission scanning electron microscopy (FE-SEM). Alterations of the methods, including different hemin and reagent concentrations, reaction time, rapid acid addition, and higher temperature (80 to 90 °C), during the annealing stage resulted in varying crystal sizes. Samples of synthesized crystals of defined size (in the range of 0.37 to 3.98 μm) were deposited and sealed between two glass coverslips before imaging.
Acid precipitation method Crystal size estimation Crystalline hemin (50 mg, 0.8 mM), from Fluka Chimie, Buchs, CH-9471 Switzerland, was dissolved in 10 mL of oxygen free 0.1 M sodium hydroxide contained in a foiled flask. Propionic acid (0.4 mL) was then added dropwise, stirring at 200 rpm, over 20 min. The black mixture was allowed to anneal at 70 °C for 18 h. Three sodium bicarbonate (0.1 M) washes, alternating with water washes, removed the amorphous aggregates. Finally, three washes with HPLC grade methanol alternating with water completed the washing procedure. The solid samples were then dried overnight over phosphorus pentoxide yielding crystalline materials in the 0.8–1.2 μm size range.
The size of the synthesized hematin anhydride crystals was estimated as follows: scanning electron microscopy images were acquired using a Hitachi S-4700 FE-SEM. The samples were coated with Au/Pd to a uniform 4-Å thickness prior to visualization at 2 kV and 10 μA. Multiple fields were used for determination of the statistical mean in size and variation. Preparations usually give monodisperse crystallite size distributions of relatively thin parallelepipeds. Within a given sample, variations of ±0.3 μm in the long dimension were measured. Image analysis
Anhydrous base-catalyzed method This was carried out under an inert atmosphere, and 2,6Lutidine (0.5 mL, 4.3 mM), dimethyl sulfoxide (5.0 mL, 70 mM), and methanol (5.0 mL, 123 mM) were sequentially added to crystalline hemin (25 mg, 0.4 mM) in a foiled flask and stirred at room temperature. The crystals were washed using the same procedure as with the acid precipitation method and dried overnight over phosphorus pentoxide. With this method, the crystalline materials were in the size range of 2 to 4 μm. Acetone preparation method In the acetone preparation method, hematin (40 mg, 63 μM) from Sigma-Aldrich, H3281, was dissolved in acetone (200 mL) in a foiled flask under stirring for 12 h at room temperature. To this solution, a 2 % sodium dodecyl sulfate solution (50 mL) over a period of 36 h was added. The crystals were washed using the same procedure as with the acid precipitation method and dried overnight over phosphorus pentoxide. The crystalline materials synthesized by this method were in few micrometers to hundreds of nanometer size range. In addition, amorphous materials were also synthesized by a method that is an alteration of the acid catalyzed method, where the materials were synthesized under no nitrogen atmosphere and the propionic acid was added in one step only. All hematin anhydride samples were
The image analysis was done by using MATLAB with custom written codes. The average THG S/N ratio was calculated as follows: In each image frame, an intensity threshold value was set for classification of the resolved hemozoin crystals above background noise. The corresponding peak intensities of detected crystals above the threshold were averaged as signal, and the intensity values lower than the threshold (for pixels where there were no crystals) were considered as noise. We used the expression S/N=(−)/(SD noise), to estimate the S/N ratio value (averaged over all crystals) for each image frame, and then it, was again averaged over the 15 sequential image frames (where < > indicates the mean and SD the standard deviation). Origin software was used for linear regression and cubic dependency analysis. For the synthesized hematin anhydride crystals, ImageJ software was used to measure the crystal dimensions from the SEM images.
Results and discussion THG emission dependence on excitation laser wavelength To determine the optimal wavelength for THG imaging of hemozoin, we carried out an excitation wavelength dependence study. Figure 1(a–c) shows images of THG emission (blue; single scan) from natural hemozoin crystals imaged in infected red blood cells (TP autofluorescence in red; 15
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Fig. 1 Combined overlay images of THG (bright crystals in blue) and TP autofluorescence (oval cells in red) of natural hemozoin crystals and red blood cells (infected with FCR-3 P. falciparum), respectively, collected at different laser excitation wavelengths (λexc =1,145 nm (a), 1,170 nm (b), and 1,245 nm (c)). The THG image is acquired in only one scan per frame with a pixel dwell time of 5 μs while the TP image was the average of 15 sequential frame scans. (d) Plot of the average
THG S/N ratio vs. λTHG (=1/3(λexc)) for the hemozoin in malariainfected cells (incident laser power=100 mW). The error bars are the calculated standard deviation from 15 sequential frame scans. The solid blue line shows the relevant portion of the linear absorption spectrum of hematin anhydride in KBr suspension. (e) Linear absorption spectrum of hematin anhydride in KBr suspension
scans averaged) with laser excitation wavelengths of 1,145 nm (a), 1,170 nm (b), and 1,245 nm (c) (images 500×500 pixels with pixel size 0.18 μm). Image analysis was performed as outlined in Image analysis section. The average THG S/N ratios for the images are plotted as a function of the fundamental excitation wavelength divided by 3 in Fig. 1(d). The S/N ratio values at each wavelength are corrected with respect to the transmission curve of the cutoff filter used in each case. It is clear that an excitation wavelength of 1,170 nm (i.e., 390 nm THG wavelength) optimizes the S/N ratio for imaging detection of hemozoin in the infected red blood cells. To investigate the basis for having a maximum THG signal at 1,170 nm excitation, first we compared the S/N ratio results with the linear absorption spectrum of the hematin anhydride (synthetic analog of natural hemozoin) in potassium bromide (KBr) suspension as shown in Fig. 1(d) and Fig. 1(e). Hemozoin is composed of a close packed condensed phase of iron(III) protoporphyrin IX rings, which form an extensive polarizable π-electron system, and it is clear from the linear absorption spectrum that it has a broad Soret band absorption from 300 to 490 nm, with peak absorption centered around 408 nm because of the π–π interaction between the porphyrin planes of the molecules within the crystalline packing. In addition,
from 380 to 420 nm (the range of THG working wavelengths in this study), the absorption profile is very broad (as shown in Fig. 1(d)) and the difference in absorbance values of these working THG wavelengths with respect to the peak of the absorption at 408 nm is not significant. For example, there is only about 7 % difference at 380 nm, approximately 3 % difference at 390 nm, and a difference of 5 % at 420 nm. It suggests that contributions from the near resonant or resonant THG processes in all these working wavelengths cannot be ignored since these processes also can efficiently produce THG signal. However, under certain conditions (for example in large crystals or clusters imaged with long excitation exposure times), we observe some attenuation of the THG signal (see Signal attenuation of THG emission section). which we believe is due to linear absorption. These results raise the question as to why the THG signal is only optimized at 1,170 nm excitation wavelength, when the range of excitation wavelengths used would produce comparable near resonant or resonant THG because of their similar absorbance values. The answer is most likely related to the exact nature of the crystals and satisfying of the phase-matching condition under different excitation wavelengths. This would make sense if the molecular structure and composition of the hemozoin crystals result in different physical anisotropic
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structures in the natural environment which are birefringent (we verified this with our polarization studies in THG emission dependence on excitation laser polarization section). It is probable that some of the favorable birefringent anisotropic structures strongly satisfy the phase-matching condition (i.e., some of the frequency components of the THG signal are phase matched with the excitation pulse frequency components and hence have equal phase velocities) by natural angle tuning (the hemozoin crystals are randomly dispersed in the infected blood serum with different orientations, hence we already inherently have the angle tuning to generate efficient THG) at that particular wavelength; therefore, the S/N ratio is optimized at 1,170 nm excitation. The contribution of instrumental artifacts due to temporal change of the pulse width at different excitation wavelengths cannot be completely ignored; however, these variations should be minor as compared to the phasematching signal output. The histogram of the THG S/N ratio values is asymmetric about the peak, 1,170 nm, with a tail extending toward longer wavelengths. This asymmetric distribution of THG signal response is attributed to the offset of excitation pulse frequencies with the phase-matched pulse frequency components with unequal phase velocities.
Partial volume effects (due to a loss in accuracy for the microscopic measurement of object volumes as the object approaches the resolution limit set by the optical point spread function (PSF)) are anticipated as we move to sub-PSF size crystals (which produce image spot sizes that are roughly the size of the PSF) from larger crystals where the spot sizes are naturally larger than the PSF and reflect the actual crystal dimensions. The THG images of crystals of three different sizes are shown in Fig. 2(a–c). Again, we believe that as a result of phase matching by angle tuning (the synthesized crystals were dispersed on the coverslip randomly and hence naturally result in angle tuning as discussed in THG emission dependence on excitation laser wavelength section), we could observe the generation of THG signal from these synthesized crystals. Phase matching is necessary for the generation of THG signal in crystals larger than the excitation wavelength, but not for the smaller crystals. The THG S/N ratio varied by approximately a factor of 10 for the crystals differing in size by an order of magnitude (Fig. 2(d)). However, the THG response is not linear over the entire range of crystal size. The linear dependency on the THG signal was observed up to a crystal size of 0.63 μm and then it began to plateau for larger size crystals. This transition boundary coincides with the expected size transition for the nonlinear PSF for the excitation conditions employed (i.e., here the estimated 3D PSF volume of our nonlinear imaging system is 0.6 μm3 corresponding to radii of 0.29 and 1.2 μm in the lateral and axial directions, respectively). The tailing off of the S/N for larger crystals is expected since the S/N is calculated as a mean of peak intensities, and the integrated signal is expected to be
THG emission dependence on synthesized hematin anhydride crystal size We next performed THG imaging with 1,170 nm excitation of synthesized crystals of hematin anhydride ranging in dimensions from ∼0.37 to 3.98 μm in order to systematically study the nonlinear response as a function of crystal size. Fig. 2 THG images (one scan per frame) of synthetic hematin anhydride crystals with different lengths ((a) ∼0.37 μm, (b) ∼1 μm, and (c) ∼3.98 μm at λexc =1,170 nm). (d) The average THG S/N ratio plot for the hematin anhydride as a function of crystal length (incident power is 100 mW and pixel dwell time is 5 μs). The inset shows the linear regression plot that extrapolates to a detection limit of 0.23 μm and quantification limit of 0.25 μm for crystal size
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approximately equal for any large crystal that completely fills the PSF. Next, to estimate the analytical detection limit (S/N ratio=3:1) as well as the quantification limit (S/N ratio=5:1) of our nonlinear imaging system, we performed a linear regression plot of THG S/N ratio response for crystals in the size range ∼0.37 to ∼0.63 μm where the variation was linear. This yielded a detection limit size of 0.23 μm and a quantification limit size of 0.25 μm, which match the theoretical diffraction limit for imaging on our system with the collection conditions employed. Crystals smaller than this limit could in principle be detected and densities measured by fluctuation analysis of the images; however, the crystals would appear in the images as diffraction limited sized spots. A recent synchrotron X-ray structural study detected a range of sizes of hemozoin crystals as both clustered clumps and individual single crystals within a single Plasmodium-infected red blood cell and at least 80 % of the crystallites present in these intact digestive vacuoles are above this detection limit and would thus be anticipated to generate a THG signal [21]. Moreover, the intracellular polydispersity persists throughout all growth stages in the blood cell. Characterizing the size dependence of the signalto-noise ratio for THG imaging of hemozoin crystals that we have observed is therefore critical for studies of infected cells which may have variable polydispersity in the crystals within the parasites. These values are important parameters for designing future studies to determine the detection limits for the onset of hemozoin production as a function of life cycle stage or time post-infection. Fig. 3 THG images (one scan per frame) of the natural hemozoin crystals (FCR-3 P. falciparum, pixel dwell time 5 μs) collected with different laser pulse energies ((a) ∼0.74 nJ, (b) ∼1.49 nJ, and (c) ∼2.37 nJ). The pixel dwell time is 5 μs. (d) Log–log plots of the excitation laser pulse energy vs. the THG signal in case of the natural (blue) and synthetic (green) hemozoin crystals. (e) The average THG S/N ratio vs. laser excitation energy plot for the natural (blue) and synthesized (green) hemozoin crystals. The natural hemozoin crystals fit to the linear regression plot that extrapolates to a detection limit of 0.21 nJ and quantification limit of 0.23 nJ energy per pulse. Similarly, the synthetic crystals fit to a cubic expression with a dependency value of 3.1±0.1
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THG emission dependence on laser pulse energy To verify the nature of the nonlinear signal as well as to estimate the detection and quantification limits in terms of excitation laser pulse energy, we imaged the THG emission of natural hemozoin and synthesized hematin anhydride crystals (size ∼0.46 μm) as a function of different excitation laser pulse energy at 1,170 nm. Figure 3(a–c) shows the images of the natural hemozoin crystals at three selected pulse energies. Here, we observed that the nonlinear signal has cubic dependency on the excitation laser pulse energy in both cases. As shown in Fig. 3(d), slopes of log–log plots of the excitation laser pulse energy vs. the nonlinear signal yielded values of 2.78±0.15 and 2.90±0.24 with linear regression in case of the natural (data shown in blue) and synthesized (data shown in green) crystals, respectively, that confirmed the signal is consistent with THG emission. Furthermore, we also estimated the S/N ratio values in case of natural and synthesized crystals as a function of excitation laser pulse energy. Figure 3(e) shows the average S/N plot. Here, we measured the uninfected blood cells with no crystals as well as empty image regions to determine the noise contribution in the S/N ratio calculation for the natural hemozoin samples. In the case of the synthesized hematin anhydride crystals, we treated only the empty image regions containing no crystals as noise. Figure 3(e) shows that the S/N ratio values of synthesized crystals (data shown in green) follow a nonlinear trend and we quantified this by fitting the data with a cubic expression. As expected for a
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THG signal, we measured a dependency value of 3.1±0.1 for the synthesized hematin anhydride crystals. In contrast, for the natural hemozoin crystals in blood cells (data shown in blue), we observed that the S/N ratio is linearly dependent on the excitation laser power over the range measured. This linear relationship is consistent with a THG signal arising from hemozoin crystals with background noise arising from THG response of the heme moiety in hemoglobin from uninfected cells and a weaker linear noise contribution from the empty image regions with no blood cells and crystals (lysed red blood cells can lead to free hemoglobin in culture, especially in the absence of in vivo clearance mechanisms). The inset of Fig. 3(e) shows the noise vs. laser pulse energy plot for reference. As explained above, we observed that the noise from the blood cell samples (data shown in purple) follows a cubic dependency (fitting line in red yields a fit exponent of 2.93±0.25), but the noise from the empty image regions (data shown in black) linearly increases with the laser pulse energy. THG emission from hemoglobin in red blood cells has been previously imaged, but at wavelengths of 1,560 and 1,400 nm [15, 22], the same cubic dependency was observed. Further, to estimate the detection and quantification limits, we analyzed the S/N ratio data with linear regression for the natural hemozoin crystals as shown in Fig. 3(e). The extrapolation of the linear regression plot gave a detection limit of 0.21 nJ (i.e., 16.28 mW incident power)
and quantification limit of 0.23 nJ (i.e., 17.84 mW incident power) energy per pulse. Here, we conclude that these are the minimum excitation energy values required to detect and quantify hemozoin crystals by THG imaging at the trophozoite stage of the malaria life cycle for our optical setup. Prior work [23] has demonstrated the stability of hemozoin crystals under measurement conditions with higher excitation energy photons. The photostability under pulsed excitation is explored further in Signal attenuation of THG emission section.
Fig. 4 Images of THG from natural hemozoin crystals (blue, one scan per frame (a, b)) and TP autofluorescence (oval cells in red, one scan per frame (c, d)) from infected red blood cells, for different excitation laser polarizations (horizontal (a, c) and vertical (b, d)). The red arrows indicate the particular crystals imaged in (a) but not in (b). The green arrows indicate the direction of the incident laser polarization. The incident power is 100 mW and pixel dwell time is 5 μs
THG emission dependence on excitation laser polarization In nonlinear microscopy, the excitation laser polarization can play an important role depending on the material properties of the sample and imaging modality employed [22]. The polarization dependence of THG emission has been seen in porous glass [24], in a multimode photonic crystal fiber [25], in ZnSe nanowires [26], and in human cornea [27]. Here, we have imaged the THG emission from the natural hemozoin crystals under different excitation laser polarizations. Figure 4 shows both THG (horizontal (a) and vertical (b)) and TP autofluorescence (horizontal (c) and vertical (d)) images for linear laser excitation polarizations. To study the effect of laser polarization on THG imaging of the malaria pigment, we compared the peak pixel intensity values (I) of the
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hemozoin crystals both in Fig. 4(a, b). The values are plotted in Fig. 5(a) for the 50 distinct crystals that could be resolved in the image. Figure 5(a) shows that there is a change in THG emission for some of the hemozoin crystals when the excitation laser polarization is changed from the horizontal to vertical direction. We used the anisotropy equation (Ihorizontal −Ivertical)/(Ihorizontal +2Ivertical) to calculate the anisotropy values for the hemozoin crystals and the absolute values are shown in Fig. 5(b). From these values, we conclude that majority of these crystals possess an anisotropic orientation and the THG is attributed to the refractive index inhomogeneities of the crystals. Nevertheless, we expect that there could be some contributions to the anisotropy from partial volume effects described in THG emission dependence on synthesized hematin anhydride crystal size section for some of the crystals in the image. In addition, there could also be contributions due to birefringent crystals that eventually have contributions from the bulk as proposed by Oron et al. [28, 29]. Confirmation of this would require experiments performed under different excitation polarization angles as proposed by Pillai et al. [30]; however, this is not possible in our case because the natural crystals are randomly dispersed in the sample so absolute crystal orientation cannot be determined. To confirm the bulk contribution, we have carefully examined the two images obtained under horizontal and vertical excitation laser polarization and observed that some of the crystals that are imaged under the horizontal polarization are not seen under the vertical polarization and vice versa (crystals marked with an arrow in Fig. 4(a) are not seen in Fig. 4(b) and vice versa (data not shown)). These findings confirm the bulk contribution due to crystal birefringence to our estimated anisotropy. In contrast to our findings here, Filippidis et al. [31] could not observe any polarization dependency in the THG images from the pharynx region of a wild-type C. elegans larva, even though they expected a weak dependence on the incident light polarization, since the THG modality was implemented to image interfaces. However, they did not compare the pixel Fig. 5 (a) Comparison of the peak pixel intensities of hemozoin crystals (from Fig. 4(a) in red and Fig. 4(b) in green) and red blood cells (inset of (a) from Fig. 4(c) in red and Fig. 4(d) in green). (b) Anisotropy plot vs. crystal number (from Fig. 4(a, b)) and red blood cell number (inset of (b) from Fig. 4(c, d))
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intensity values between images collected with two different excitation polarization states to quantify their results. Our results point to the fact that the observed anisotropic structure and birefringent nature of the crystals must have contributed to a very strong phase-matched THG signal output in case of the wavelength, crystal size, and laser pulse energy dependency measurements. We also show the results of peak pixel intensity values and the anisotropy values on the polarized TP autofluorescence images of the red blood cells in the insets of subpanels (a) and (b) of Fig. 5, respectively. The low calculated anisotropy values (1 μm) do exhibit significant diminution of the THG signal at the maximum exposure time
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(for the 3-μm crystals, the signal loss is ∼40 % at 0.86 nJ and 70 % at 1.05 nJ per pulse). However, for up to ∼2 min of exposure time, even the larger crystals do not show any signal loss. Previous THG imaging studies on plant samples did not show the THG signal loss as a function of time [14, 15]. In contrast, the diminution of the signal observed in our study at higher laser powers and larger crystals could be attributed to the near resonance effect (as discussed in THG emission dependence on excitation laser wavelength section) since one third of the laser excitation wavelength (i.e., ∼390 nm) is close to the peak of the Soret band absorption, 408 nm, of the hemozoin crystals. Another possibility is that the higher laser powers simply result in structural ablation of the composite crystals leading to reduced THG signal which would consistent with our measurement of lower S/N ratio for smaller crystals. However, ablation of the larger crystals or clusters would be resolvable in the image series and we did not observe any reduction of imaged crystal sizes which support the conclusion that it indeed related to linear absorption and saturation.
These measured parameters for the optimal THG response will serve as a guide for the design of studies to determine onset and progression of hemozoin crystal production during parasite life cycle as well as for potential THG imaging studies to screen for the efficacy of antimalarial drugs that target hemozoin production.
Conclusions In conclusion, we have determined the optimal THG imaging conditions for the malaria pigment; hemozoin in the trophozoite stage of the malaria life cycle as well as synthesized hematin anhydride crystals using a custom-built multimodal nonlinear laser scanning microscope. We observed that the optimal wavelength for maximizing the THG S/N ratio is 1,170 nm. The detection and quantification limits of our nonlinear imaging system for the natural hemozoin (trophozoites) crystals were extrapolated as laser powers of 16.28 and 17.84 mW, respectively. In addition, with respect to the size of the hematin anhydride crystals, the detection and quantification limits at the optimal 1,170-nm excitation wavelength were extrapolated to be 0.23 and 0.25 μm, respectively. In our previous work [11], we estimated parasitemia levels of 40 parasites/μL in 50 μL of infected blood, assuming similar collection conditions in a flow cytometry geometry with sampling over the 5-min time scale. Based on the results from the current study, we feel that it is possible to optimize the detection in a microfluidic device where hydrodynamic focusing is used to pass red blood cells single file through a slightly expanded laser focus. Under these conditions, given sufficient time in the focal volume (∼10 μs which would establish the maximum sample flow rate), single infected cells at the trophozoite stage should be detectable above the noise. The THG polarization study showed that the refractive index inhomogeneities, partial volume effects, and the effect of crystal birefringence can cause a change in THG emission from the hemozoin crystals for orthogonal laser polarizations.
Acknowledgments We thank the Natural Science Education Research Canada (NSERC) for an NSERC I2I grant and a Discovery grant to carry out these studies (PWW and EG). PWW also acknowledges funding from the Fessenden Chair in Science Innovation and the Canadian Foundation for Innovation. DSB acknowledges support from NSERC, the CRC, and the FQRNT.
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