Diamond Magnetic Microscopy of Malarial Hemozoin Nanocrystals.

Magnetic microscopy of malarial hemozoin nanocrystals is performed by optically detected magnetic resonance imaging of near-surface diamond nitrogen-vacancy centers. Hemozoin crystals are extracted from Plasmodium falciparum-infected human blood cells and studied alongside synthetic hemozoin crystals. The stray magnetic fields produced by individual crystals are imaged at room temperature as a function of the applied field up to 350 mT. More than 100 nanocrystals are analyzed, revealing the distribution of their magnetic properties. Most crystals (96%) exhibit a linear dependence of the stray-field magnitude on the applied field, confirming hemozoin's paramagnetic nature. A volume magnetic susceptibility of 3.4 × 10-4 is inferred with use of a magnetostatic model informed by correlated scanning-electron-microscopy measurements of crystal dimensions. A small fraction of nanoparticles (4/82 for Plasmodium falciparum-produced nanoparticles and 1/41 for synthetic nanoparticles) exhibit a saturation behavior consistent with superparamagnetism. Translation of this platform to the study of living Plasmodium-infected cells may shed new light on hemozoin formation dynamics and their interaction with antimalarial drugs.

INTRODUCTION

Magnetic field sensors based on diamond nitrogen-vacancy (NV) centers have emerged as a powerful platform for detecting nanomagnetism in biological samples [1,2]. With this technique, magnetic fields from magnetotactic bacteria [3], ferritin proteins [4], magnetically labeled cancer cells [5], and neuronal currents [6] have been detected with a remarkable combination of spatial resolution and sensitivity. Diamond magnetic microscopy has even been able to resolve magnetic fields produced by individual nanoparticles exhibiting ferromagnetism and superparamagnetism [7-9]. However, observation of individual paramagnetic nanoparticles at ambient temperature has remained a challenge, owing to their weaker magnetic signatures.

Of particular interest are paramagnetic hemozoin biocrystals that nucleate inside several blood-feeding organisms [10], including the Plasmodium species responsible for malaria. Malarial parasites feed on their host’s hemoglobin for essential amino acids, while decomposing the iron complexes into highly toxic free radicals. These radicals are subsequently bound into chemically inert elongated crystals (50–1500 nm in size) called “hemozoin” [10-12]. Hemozoin crystals are a biomarker for malaria, and a substantial effort has been devoted to developing diagnostic platforms based on their detection [10,13]. Hemozoin detection is also used in pharmacological studies of malaria [14], since some antimalarial drugs work by altering hemozoin formation [11,12].

Hemozoin crystals have characteristic optical properties, including birefringence [15], linear dichroism [16], and nonlinear dielectric susceptibility [17], that allow their detection without the use of extrinsic labels. They are also paramagnetic due to the presence of unpaired electrons in their Fe3+ centers, meaning they are magnetized only in the presence of an external magnetic field. Direct detection of hemozoin’s magnetization is intriguing because it gives quantitative information related to crystal size and iron composition.

Several methods of studying hemozoin magnetic signatures have been demonstrated, including magneto-optical rotation [18-20], nuclear-magnetic-resonance relaxometry [21,22], electron paramagnetic resonance [23], and magnetic separation [24]. Direct magnetic detection of hemozoin ensembles has been performed by bulk magnetometry [25-27]. However, detection of stray magnetic fields produced by single hemozoin nanocrystals has not yet been reported, likely due to stringent requirements on sensitivity and spatial resolution.

We hypothesized that diamond magnetic microscopy [3,5,9,28,29] possesses sufficient sensitivity and spatial resolution to image the stray magnetic field produced by individual hemozoin nanocrystals. To test this, we performed room-temperature optically-detected-magnetic-resonance (ODMR) imaging of a NV-doped diamond substrate in contact with either “natural” (Plasmodium-produced) or synthetic hemozoin nanocrystals. Spatially resolved maps of the magnetic fields produced by individual hemozoin nanocrystals were obtained and used to characterize the distribution of their paramagnetic properties. With appropriate modifications, this detection strategy may be used to study the formation dynamics of hemozoin crystals in living Plasmodium-infected cells.

DETECTION PRINCIPLE

NV centers are spin-1 defects in the diamond lattice. The energy levels and optical excitation and emission path-ways of the NV center are shown in Fig. 1(a). For a magnetic field B‖ applied along the NV symmetry axis, the |0〉 ↔ | ± 1〉 spin transition frequencies are f ± D ± γNVB‖, where D = 2.87 GHz is the zero-field splitting and γNV = 28 GHz/T is the NV gyromagnetic ratio [Fig. 1(b)]. The principle of NV magnetometry is to measure these transition frequencies precisely with ODMR techniques [1,2]. When 532-nm light continuously excites NV centers, the ground-state population is optically polarized into |0〉 via a nonradiative, spin-selective decay path-way involving intermediate singlet states. Because of the same spin-selective decay mechanism, NV centers excited from |0〉 emit fluorescence (collected at 650–800 nm) at a higher rate than those originating from |1±〉. Application of a transverse microwave magnetic field mixes the spin populations, resulting in a dip in fluorescence when the microwave frequency matches the spin transition frequencies, f± [Fig. 1(c)]. Monitoring of NV fluorescence as the microwave frequency is swept across the resonances reveals f± and thus B‖.

FIG. 1.

Diamond magnetic microscopy. (a) NV energy-level diagram depicting magnetic sublevels (|0〉, 1), optical (green arrow), fluorescence (red arrows), and nonradiative (gray arrows) pathways. Gray arrows excitation show spin-selective intersystem crossing leading to polarization into the |0〉 ground-state sublevel. (b) Energy splitting of the ground-state sublevels in an external magnetic field applied along the NV axis. Blue arrows indicate the f± microwave transitions. (c) Example of an ODMR spectrum. From the separation between peaks, the projection of the local magnetic field along the NV axis is inferred. (d) Epifluorescence ODMR microscope used for magnetic imaging. Dry hemozoin crystals are placed on top of a diamond substrate with an ~ 0.2-μm top layer doped with NV centers. (e) Experimental and photoelectron-shot-noise-limited detection threshold for 65 × 65 nm2 detection pixels versus averaging time. The experimental data are fit to the function with α = 8.4 ± 0.1 μT s1

The minimum detectable magnetic field of a Lorentzian ODMR signal is limited by photoelectron shot noise as [2,30,31] where Γ is the full-width-at-half-maximum (FWHM) linewidth, C is the contrast (the relative difference in ODMR signal on and off resonance), I0 is the photoelectron detection rate, and t is the measurement time. In room-temperature NV ODMR experiments, the spin projection noise is typically more than one order of magnitude lower than the photoelectron shot noise and is therefore negligible [32-34]. With typical experimental values (Γ = 12 MHz, C = 0.02, I0 5 106 electrons/s) for a 65 × 65 nm2 pixel, detection Eq.= (1) predicts Bmin ≃ 7.4 μT for t = 1 s. Experimentally, we observe a detection threshold that is within 15% of the photoelectron shot-noise limit [Fig. 1(e)]. The experimentally determined Bmin is calculated as the standard deviation of field values in 65 × 65 nm2 pixels in magnetic images lacking visible features. Scaling of the experimental detection threshold to a 390 × 390 nm2 pixel (approximately the diffraction-limited resolution), gives Bmin ≃ 1.4 μT threshold for t = 1 s. This detection is a factor of six lower than that for 65 × 65 nm2 pixels because I0 increases by a factor of 36 [Eq. (1)].

This detection threshold is sufficient to image the microtesla fields from hemozoin nanocrystals. In our experimental geometry [Fig. 1(d)], hemozoin crystals are magnetized along the x axis and the B component of the magnetic field is detected by diamond magnetic microscopy. In the point-dipole approximation [35], which is valid for crystals with dimensions smaller than the ~390-nm spatial resolution of our microscope, the magnetic field produced from a single crystal (volume V) is where B0 is the applied magnetic field and χ is the volume magnetic susceptibility. χ depends on the crystal orientation and purity, and values of (3.2−4.6) 10−4 have been reported in the literature [10,18]. For ×conservative values χ = 3.2 × 10−4, V = 100 × 100 × 100 nm3, and NV sensing depth z = 200 nm, the field produced by a hemozoin nanocrystal in a B0 = 350 mT applied field has a minimum of Bx= −1.1 μT at x = y = 0 and maxima of B = 0.2 μT at y = 0, x = ± 245 nm.

To more accurately describe the instrument response, magnetostatic modeling is used to calculate the B(x, y, z) field produced by elongated crystals, with dimensions taken from scanning-electron-microscopy (SEM) images. We then integrate B over the NV vertical distribution, which is assumed to be uniform from 20 to 220 nm below the diamond surface (Appendix B). Finally, we account for the effect of optical diffraction and image drift by convolution with a two-dimensional Gaussian kernel (“blur”) with 540-nm FWHM. A discussion of model parameters is given in Appendix F.

EXPERIMENTAL METHODS

Figure 1(d) depicts the epifluorescence ODMR micro-scope used for diamond magnetic microscopy. The diamond substrate is a [110]-polished, 2 × 2 × 0.08 mm3 type-Ib-diamond substrate grown by high-pressure, high-temperature synthesis. 4He+ ions were implanted into the substrate [31] at three different energies (5, 15, and 33 keV) to produce a roughly uniform distribution of vacancies in an ~ 200-nm near-surface layer (see Appendix B). After implantation, the diamond was annealed in a vacuum furnace [36] at 800 °C (4 h) and 1100 °C (2 h) to produce a near-surface layer of NV centers with a density of ~ 10 ppm. Microwaves are delivered by copper loops printed on a contacting glass coverslip. A magnetic field, , produced by a pair of permanent magnets, points along one of the in-plane NV axes. A linearly polarized 532-nm laser beam (0.2 W) excites the NV centers over an area of ~ 40 × 40 μm2, and their fluorescence is imaged onto a scientific CMOS (sCMOS) camera. A detailed description of the apparatus is given in Appendix B.

Magnetic field maps are obtained by performing ODMR imaging of the near-surface NV layer (see Appendix D). Fluorescence images (600 pixels 600 pixels, 39 × 39 μm2 field of view, 3-ms exposure time) of the NV layer are recorded at 16 different microwave frequencies around each of the NV ODMR resonances (32 frequencies in total). The sequence is repeated, and the image set is integrated for several minutes to increase the signal-to-noise ratio. The fluorescence-intensity-versus-microwave-frequency data for each pixel are fit by Lorentzian functions to determine the ODMR central frequencies, f±. The magnetic field projection along the NV axis is then calculated as B‖ ≡ B0 +B = (f+ − f−)/(2γNV). Determining B‖ in this way eliminates common-mode shifts in f± due to temperature-dependent changes in zero-field splitting D [37] and variations in longitudinal strain [33,38]. The external field, B0, is subtracted from the image, revealing a map of the stray magnetic fields, B, produced by the magnetized nanocrystals.

Figure 2 shows SEM images of the synthetic and natural hemozoin nanocrystals studied here. The synthetically produced hemozoin crystals (tlrl-hz, InvivoGen) have an elongated shape and varied dimensions (50–1500 nm). The natural hemozoin nanocrystals, extracted from human red blood cells cocultured with Plasmodium falciparum [24], have similar elongated shapes with slightly larger average dimensions. For diamond magnetic microscopy, hemozoin samples are diluted in water and drop-cast on the diamond surface to yield a relatively nonaggregated surface density with approximately 0.04 nanocrystals/μm2.

FIG. 2.

Hemozoin nanocrystals. (a) SEM image of synthetic hemozoin nanocrystals on a diamond substrate. (b) SEM image of natural hemozoin nanocrystals extracted from human red blood cells cocultured with Plasmodium falciparum, on a diamond substrate. (c) Molecular structure of a hematin dimer, which can form hemozoin crystals when joined together by hydrogen bonds. The molecular structure is expected to be the same for natural and synthetic hemozoin crystals.

RESULTS

Figure 3(a) shows a bright-field transmission image of natural hemozoin crystals dispersed on the diamond sensor’s surface. Most of the bright features in the image come from host-cell residue; they do not appear in magnetic images. Results from another region without residue are reported in Appendix 3.

FIG. 3.

Natural hemozoin. (a) Bright-field transmission image of natural hemozoin crystals dispersed on a diamond substrate. The scale bar is the relative transmission. (b) SEM image of the same nanocrystals. To enhance hemozoin visibility, the SEM image is modified by a segmentation procedure, depicted in Fig. 7. (c) Diamond-magnetic-microscopy image for applied field B0 = 350 mT. Nanocrystals labeled n1–n5 are studied in detail in Figs. 4(a)–4(e).

Figure 3(b) displays a modified SEM image of the same region as in Fig. 3(a). We use an image-segmentation procedure (Appendix C) to more clearly visualize the hemozoin crystals. Figure 3(c) shows the corresponding magnetic field map obtained by diamond magnetic microscopy. Of a total of 120 features identified as potential hemozoin nanocrystals in the SEM image, 82 exhibit magnetic features resolved by our technique. Surprisingly, the two magnetic features with the largest magnitude correspond to crystals with dimensions of ~200 nm or less in the SEM image. Such anomalously strong features are observed consistently, but infrequently (less than 5% of all magnetic features), in both natural and synthetic hemozoin samples. We tentatively attribute them to superparamagnetism [39], for reasons discussed below.

Five example crystals are labeled n1–n5 in the images in Figs. (b) and 3(c), including one of the aforementioned strongly magnetized nanocrystals (n5). Figure 4(a) shows SEM images of each example crystal; n1–n4 have a typical size and shape for these crystals, whereas n5 is barely visible, with dimensions of < 200 nm. Figure 4(b) shows the corresponding diamond-magnetic-microscopy images taken at B0 = 186 mT. Each crystal exhibits a different field pattern characteristic of its unique size, shape, and orientation.

FIG. 4.

Magnetic imaging of individual natural hemozoin nanocrystals. (a) SEM images of hemozoin nanocrystals n1–n5, labeled in Fig. 3. (b) Corresponding diamond-magnetic-microscopy images (B0 = 186 mT) for each crystal. (c) Simulated magnetic images (χ = 3.4 × 10−4, B0 = 186 mT) obtained with nanocrystal dimensions inferred from (a). Only the crystal at the center of each SEM image is included in the model. (d) Line cuts of each nanocrystal (red, measured; gray, simulated), from which the field-pattern amplitude ΔB is inferred. A minimum feature width of ~ 400-nm FWHM is observed for n5, close to the optical diffraction-limited resolution of our microscope. (e) ΔB(B0) = 0 (zero-coercivity assumption). (f) Histogram of ΔB (B0 = 350 mT) of the 78 crystals exhibiting linear paramagnetic behavior (including n1–n4). The four crystals exhibiting superparamagnetic behavior (including n5) are excluded from the analysis. (g) Fitted slopes, dΔB/dB0, as a function of crystal area, A, as determined from SEM images. The data are fitted with an empirical saturation function, dΔB/dB0 = Smax/(1 + Asat/A), with Smax = 16.5 ± 1.4 μT/T and Asat = 0.17 ± 0.03 μm2.

Figure 4(c) shows the expected magnetic field patterns of each nanocrystal, calculated with the procedure described in Sec. II. Each nanocrystal is modeled as a three-dimensional (3D) ellipsoid with uniform susceptibility and dimensions inferred from the corresponding SEM images. The height of the crystals is assumed to be 200 nm. For n1–n4, the model produces field patterns similar to those observed experimentally. The pattern amplitude is best described with a volume magnetization M = 50 A/m, which corresponds to a volume susceptibility χ = μ0M/B0 = 3.4 × 10−4, where μ0 = 4π × 10−7 m T/A is the vacuum permeability. This value of χ is well within the range of literature values for hemozoin [10,18] and is in good agreement for most crystals, despite a wide variation in their magnetic patterns. This demonstrates that crystal size, shape, and orientation are the primary factors in determining the magnetic pattern behavior. Factors contributing to ~ 25% or less uncertainty in χ are discussed in Appendix F.

For crystal n5, however, the model does a poor job of describing the magnetic behavior. It predicts a field-pattern amplitude more than one order of magnitude lower than the observed value, suggesting n5 is not paramagnetic hemozoin. It is difficult to estimate this particle’s magnetization, since it may arise from a small inclusion or an adjacent particle not resolved in the SEM image.

Line cuts of the magnetic field patterns for n1–n5 are shown in Fig. 4(d). The line cuts are obtained by averaging B values over six rows (390 nm) in a band along the magnetic feature, as indicated in Figs. 4(b) and 4(c). The line cuts are used to calculate the magnetic pattern amplitude ΔB, determined from the difference in extreme B values in the line cuts. The uncertainty in ΔB is approximately ±0.2 μT, on the basis of the scatter in the B values in the line cuts in a magnetic-feature-free region. The amplitudes for n1–n4 differ due to size and shape, but all fall in the range ΔB = 1.3−2.9 μT. However, the amplitude for n5 is much larger, ΔB = 11.0 ± 0.2 μT. The FWHM of this feature is ~400 nm, close to our microscope’s diffraction-limited spatial resolution, providing further evidence that it comes from a pointlike particle.

Magnetic images of all nanocrystals shown in Fig. 3 were obtained for six values of the external field: B0 = 83, 122, 186, 240, 300, and 350 mT. From these images, we identify 82 individual magnetized crystals and calculate ΔB as a function of B0 for each of them. The ΔB(B0) curves for all 82 crystals are provided in Fig. 13. Figure 4(e) shows the curves of nanocrystals n1–n5. A linear dependence is found for n1–n4 and is characteristic of a paramagnetic response. In total, 78 of 82 natural hemozoin crystals show a similar linear behavior, with a slope in the range dΔB/dB0 = 4−16 μT/T.

FIG. 13.

Natural hemozoin: field-dependent magnetization. ΔB(B0) curves for all 82 natural hemozoin crystals reported in Sec. IV (left). Magnetic image (B0 = 350 mT) showing the positions of each nanocrystal (right).

The remaining four nanoparticles from the natural hemozoin sample exhibit a saturation behavior, as seen for n5. These curves are fit with a Langevin function of the form ΔB(B0) = a[coth(bB0) − 1/(bB0)], where a and b are fit parameters. For n5, a = 13.3 ± 0.7 μT and b = 0.017 ± 0.002 mT−1. This model is commonly used to describe superparamagnetic nanoparticles in the limit that the thermal energy exceeds the magnetic anisotropy energy [40]. The large magnetization and saturation behavior observed in these small nanoparticles is consistent with recent reports of superparamagnetism in hemozoin samples [39,41]. However, only a small fraction (~ 5% or less) of the nanoparticles exhibit such behavior. This may explain why superparamagnetic behavior was not observed in previous ensemble studies with unrefined samples [18,22,25]. The atomic structure of these outlier nanoparticles remains a topic for future work.

A histogram of B for all 78 paramagnetic nanocrystals at B0 = 350 mT is shown in Fig. 4(f). The distribution is characterized by a mean amplitude of 2.9 μT, a median of 2.6 μT, and a standard deviation of 1.6 μT. Figure 4(f) plots the best-fit slopes, dΔB/dB0, as a function of crystal area, as determined from SEM images. The slopes increase in a roughly monotonic fashion before saturating when the crystal dimensions exceed the spatial resolution of the microscope. This behavior is expected for crystals with uniform susceptibility. For crystal volumes much smaller than the sensing voxel, V «Vsense ≈ 0.4 × 0.4 × 0.2 μm3, Eq. (2) predicts dΔB/dB0 ∝ χ V, while for V »Vsense we expect dΔB/dB0 ∝ χ independent of crystal dimensions.

We have also measured the magnetic properties of synthetic hemozoin crystals manufactured by InvivoGen. These nanocrystals are commonly used as a model for natural hemozoin, owing to their ease of procurement and nearly identical crystal morphology and chemical structure [42,43]. Previous studies reported similarities in the ensemble magnetic properties of synthetic and Plasmodium falciparum–extracted hemozoin [44]; however, here we compare the distribution of their magnetic properties at the single-nanocrystal level.

Figure 5(a) shows a confocal reflection image of dried synthetic hemozoin crystals dispersed on the diamond substrate. Of a total of 46 dark features identified as potentially being hemozoin, 41 produce magnetic features in the diamond-magnetic-microscopy images [Fig. 5(b)]. As with natural hemozoin, B(B0) curves are generated by monitoring these features in magnetic images taken at six different applied fields. The ΔB(B0) curves for all 41 nanocrystals are displayed in Fig. 14. One nanocrystal exhibits a Langevin saturation, suggesting superparamagnetic behavior, while the remaining 40 crystals exhibit a linear dependence. Figure 5(c) shows the ΔB(B0) curves for three example nanocrystals exhibiting linear behavior, labeled s1–s3 in Fig. 5(b). Magnetostatic modeling [Fig. 10(d)] of these three nanocrystals shows good agreement with the experimental images with χ = 3.4 × 10−4. This is the same value as found for natural hemozoin [Figs. 4(c) and 4(d)], indicating the natural and synthetic crystals have similar magnetic properties.

FIG. 5.

Synthetic hemozoin. (a) Confocal reflection image (excitation at 405 nm) of synthetic hemozoin nanocrystals on a diamond substrate. In total, 46 dark features are identified as likely hemozoin nanocrystals. (b) Corresponding diamond-magnetic-microscopy image at B0 = 350 mT. 41 of 46 possible crystals exhibit an observable magnetic feature. (c) ΔB(B0) curves for three synthetic hemozoin crystals labeled in (b). Solid lines are weighted linear fits. (d) Histograms of dΔB/dB0 for natural and synthetic hemozoin crystals.

FIG. 14.

Synthetic hemozoin: field-dependent magnetization. ΔB(B0) curves for all 41 synthetic hemozoin crystals reported in Sec. IV (left). Magnetic image (B0 = 350 mT) showing the positions of each nanocrystal (right).

FIG. 10.

Individual synthetic hemozoin crystals. (a) Confocal reflection images of hemozoin nanocrystals s1–s3, labeled in Figs. 5(a) and 5(b). The estimated area used for magneto-static modeling is outlined in red. (b) Corresponding diamond-magnetic-microscopy images (B0 = 350 mT) for each crystal.(c) Simulated magnetic images (χ = 3.4 × 10−4, B0 = 350 mT)obtained with nanocrystal dimensions roughly estimated from(a). (d) Line cuts of each nanocrystal (red, measured; gray, simulated), from which the field pattern amplitude ΔB is inferred.

Figure 5(d) shows histograms of the fitted slopes, dΔB/dB0, for all 40 paramagnetic synthetic crystals and all 78 paramagnetic natural crystals. The synthetic nanocrystals have a slightly smaller slope (mean 5.5, median 4.3 μT/T) than natural hemozoin (mean 8.1, median 7.2 μT/T), while the standard deviation is similar (6.2 and 8.0 μT/T, respectively). This is likely due to a small difference in their size distributions (see Appendix C and Fig. 8).

FIG. 8.

Size comparison of natural and synthetic hemozoin crystals. (a) SEM image of synthetic hemozoin crystals dispersed on a diamond substrate. (b) Histogram of crystal area for natural and synthetic hemozoin crystals, as determined from SEM images in Figs. 7(a)–7(c) and 8(a).

OUTLOOK AND CONCLUSION

Our results demonstrate the capability of diamond magnetic microscopy to simultaneously measure the magnetic properties of numerous individual biocrystals. Whereas bulk measurements yield ensemble-average properties, diamond magnetic microscopy can measure the distribution of nanocrystal susceptibilities, extract information about each crystal’s dimensions and orientation, and differentiate paramagnetic hemozoin from superparamagnetic nanoparticles. This brings up the intriguing possibility of using this platform to monitor the formation dynamics of individual hemozoin nanocrystals in living cells without the use of extrinsic contrast agents.

To assess the feasibility of performing magnetic imaging of hemozoin inside cells, we estimate a typical hemozoin crystal volume is V = 0.04 μm3, on the basis of the median area found from SEM images (0.2 μm2) and an assumed thickness of 0.2 μm. We assume the hemozoin crystal forms in a digestive vacuole within a Plasmodium-infected red blood cell (typical thickness 2 μm [13]) such that it is located 2 μm from the diamond surface. Such a crystal with susceptibility χ = 3.4 × 10−4 under an applied field B0 = 350 mT would produce a magnetic pattern amplitude at the diamond surface (z = 2 μm) of ΔB = 0.057 μT [Eq. (2)]. This field shift would be detectable with a signal-to-noise ratio of one after ~ 10 min of averaging using the present diamond sensor with 390 × 390 nm2 detection pixels (see Sec. II). However, the 200-nm NV layer currently used is optimized for negligible diamond-hemozoin separation. If the separation is ~ 2 μm, an NV layer of 1 μm would be more optimal, leading to a signal-to-noise ratio of one after approximately 2 min. If a higher magnetic field is used (already realized for nanoscale NV magnetometry [45,46]), a signal-to-noise ratio of one should be obtained for B0 = 3 T after ~ 1.6 s of signal averaging.

This sensitivity would be suffcient to monitor the formation dynamics [47] of individual hemozoin nanocrystals on minute-to-hour timescales. It could enable monitoring of crystal position and orientation throughout the parasite life cycle under different conditions, including the influence of antimalarial drugs. However, the movement of red blood cells would need to be constrained such that crystals remain close to the diamond surface. This may be accomplished by promotion of cell adhesion [48] or sedimentation [49], confinement of cells within microstructures [36,50], and/or increase of the viscosity of the surrounding medium [51-53]. If the platform is simplified and miniaturized, it may also find application as a label-free malaria diagnostic tool with sensitivity rivaling the current staining or microscopy standard [18]. Our platform can also be used to study paramagnetic substances other than hemozoin. Figure 11 shows diamond-magnetic-microscopy images of the pharmacological agent hemin.

FIG. 11.

Magnetic imaging of hemin crystals. (a) Bright field transmission image of hemin crystals. (b) Corresponding diamond-magnetic-microscopy image (B0 = 186 mT). The inplane component of the applied field is labeled with an arrow. An out-of-plane component of similar magnitude is also present. Smaller particles are synthetic hemozoin crystals that are dispersed along with hemin crystals.

In summary, we use diamond magnetic micro-scopy to characterize the distribution of magnetic properties of synthetic and natural hemozoin samples at the individual nanocrystal level. More than 95% of the nanocrystals exhibit paramagnetic behavior, with magnetic field patterns well described by a magnetostatic model using a volume susceptibility χ = 3.4 × 10−4. Five of the 123 nanocrystals studied exhibit anomalously large magnetization that saturates at fields above approximately 0.1 T, suggesting superparamagnetism. Further work is needed to determine the composition and structure of these outlier nanoparticles. With improvements in the experimental setup, diamond magnetic microscopy should be capable of imaging hemozoin formation dynamics in living cells, with implications for malaria diagnosis and drug development.

TABLE I.

Methods for label-free imaging of individual hemozoin crystals.

Imaging method	Physical observables	Penetration of probe	Frame rate (Hz)
Absorption microscopy [57]	Optical absorption	Optical field	~ 100
Polarization microscopy [58]	Birefringence or dichroism	Optical field	~ 100
Differential interference contrast [59]	Refractive index	Optical field	~ 50
Resonance Raman microscopy [60]	Vibrational structure	Intense optical field	~ 1
Third-harmonic generation [17]	Crystal structure	Intense optical field	~ 1
Diamond magnetic microscopy (this study)	Magnetic moment	dc or ac magnetic field	~ 1