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 ($96\mathrm{\%}$) exhibit a linear dependence of the stray-field magnitude on the applied field, confirming hemozoin’s paramagnetic nature. A volume magnetic susceptibility of $3.4\times {10}^{-4}$ 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.

Figure 1

Diamond magnetic microscopy. (a) NV energy-level diagram depicting magnetic sublevels ($|0⟩$, $|\pm 1⟩$), optical excitation (green arrow), fluorescence (red arrows), and nonradiative (gray arrows) pathways. Gray arrows 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 $f_{\pm }$ 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-$\mu \mathrm{m}$ top layer doped with NV centers. (e) Experimental and photoelectron-shot-noise-limited detection threshold for $65\times 65{\mathrm{nm}}^2$ detection pixels versus averaging time. The experimental data are fit to the function $B_{\min }=\alpha /\sqrt{t}$, with $\alpha =8.4\pm 0.1\mu \mathrm{T}{\mathrm{s}}^{1/2}$.

Figure 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.

Figure 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 $B_0=350\mathrm{mT}$. Nanocrystals labeled n1–n5 are studied in detail in Figs. 4–4.

Figure 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 ($B_0=186$ mT) for each crystal. (c) Simulated magnetic images ($\chi =3.4\times {10}^{-4}$, $B_0=186$ 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 $\mathrm{\Delta }B$ 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) $\mathrm{\Delta }B(B_0)$ for each crystal. Solid lines are weighted fits to a line (n1–n4) or Langevin function (n5). All fits are constrained to intercept the origin, $\mathrm{\Delta }B(0)=0$ (zero-coercivity assumption). (f) Histogram of $\mathrm{\Delta }B(B_0=350\mathrm{mT})$ 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, $d\mathrm{\Delta }B/d{B}_0$, as a function of crystal area, $A$, as determined from SEM images. The data are fitted with an empirical saturation function, $d\mathrm{\Delta }B/d{B}_0=S_{\max }/(1+A_{\mathrm{sat}}/A$), with $S_{\max }=16.5\pm 1.4\mu \mathrm{T}/$T and $A_{\mathrm{sat}}=0.17\pm 0.03\mu {\mathrm{m}}^2$.

Figure 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 $B_0=350\mathrm{mT}$. 41 of 46 possible crystals exhibit an observable magnetic feature. (c) $\mathrm{\Delta }B(B_0)$ curves for three synthetic hemozoin crystals labeled in (b). Solid lines are weighted linear fits. (d) Histograms of $d\mathrm{\Delta }B/d{B}_0$ for natural and synthetic hemozoin crystals.

Figure 6

NV-layer fabrication. (a) SRIM vacancy-depth profile for implantation conditions discussed in the text. (b) Time-temperature graph for the annealing procedure used in this study.

Figure 7

SEM processing. (a) Unprocessed SEM micrograph of the natural hemozoin sample. (b) A mask used to highlight and colorize hemozoin crystals and to attenuate their background. (c) Modified SEM image as shown in Fig. 3. (d) Unprocessed and (e) modified SEM images of hemozoin crystals n1–n5 addressed in Figs. 4–4.

Figure 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. 7–7 and 8.

Figure 9

Data processing. (a) Fluorescence images are acquired at each of 32 different microwave frequencies (16 frequencies for each of the $f_{\pm }$ spin transitions). (b) For any given pixel, two 16-point ODMR curves (fluorescence intensity versus microwave frequency) are generated. Each ODMR curve is fit to a Lorentzian function to reveal the ODMR central frequencies for that pixel. This is repeated for each pixel, yielding two maps of ODMR central frequencies corresponding to $f_{+}$ and $f_{-}$. (c) The magnetic field pattern is obtained by subtraction of the images for $f_{+}$ and $f_{-}$, division by $2{\gamma }_{\mathrm{NV}}$, and subtraction of the applied $B_0$ field. (d) An example fluorescence image for a fixed microwave frequency along with a bright-field transmission image of the same field of view. (e) Intermediate step showing separate maps for frequencies $f_{+}$ and $f_{-}$. (f) The final map, taken at $B_0=350$ mT, is proportional to stray magnetic fields, ${\gamma }_{\mathrm{NV}}{B}_x=(f_{+}-f_{-})/2-{\gamma }_{\mathrm{NV}}{B}_0$. This is shown alongside a map of residual nonmagnetic shifts, $(f_{+}+f_{-})/2-2D$. Note that $f_{-}$ is defined such that it is negative when $|-1⟩$ is lower in energy than $|0⟩$. The horizontally varying pattern is an artifact of the sCMOS camera’s dual-sensor read-out. This artifact is present when rolling-shutter mode is used, but is eliminated by our subtraction procedure.

Figure 10

Individual synthetic hemozoin crystals. (a) Confocal reflection images of hemozoin nanocrystals s1–s3, labeled in Figs. 5 and 5. The estimated area used for magnetostatic modeling is outlined in red. (b) Corresponding diamond-magnetic-microscopy images ($B_0=350$ mT) for each crystal. (c) Simulated magnetic images ($\chi =3.4\times {10}^{-4}$, $B_0=350$ 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 $\mathrm{\Delta }B$ is inferred.

Figure 11

Magnetic imaging of hemin crystals. (a) Bright-field transmission image of hemin crystals. (b) Corresponding diamond-magnetic-microscopy image ($B_0=186$ mT). The in-plane 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.

Figure 12

A second region of the natural hemozoin sample. (a) Bright-field transmission image of natural hemozoin crystals dispersed on a diamond substrate. (b) SEM image of the same nanocrystals. Dark rectangles are due to overcharging during zooming. (c) Corresponding diamond-magnetic-microscopy image at $B_0=350\mathrm{mT}$ showing the positions of each nanocrystal. (d) $\mathrm{\Delta }B(B_0)$ curves for all 12 hemozoin crystals. (e) Histogram of $d\mathrm{\Delta }B/d{B}_0$ determined from the linear slopes in (e). (f) Magnified SEM and magnetic images of crystals 1–4.

Figure 13

Natural hemozoin: field-dependent magnetization. $\mathrm{\Delta }B(B_0)$ curves for all 82 natural hemozoin crystals reported in Sec. 4 (left). Magnetic image ($B_0=350\mathrm{mT}$) showing the positions of each nanocrystal (right).

Figure 14

Synthetic hemozoin: field-dependent magnetization. $\mathrm{\Delta }B(B_0)$ curves for all 41 synthetic hemozoin crystals reported in Sec. 4 (left). Magnetic image ($B_0=350\mathrm{mT}$) showing the positions of each nanocrystal (right).

Figure 15

Impact of model parameters on magnetic susceptibility $\chi$. (a) Line cuts of measured (red) and simulated (gray) magnetic patterns from crystal n4 [reproduced from Fig. 4] obtained with the initial model parameters shown on the right. (b) Simulated line cuts of n4 for different model parameters. Only one parameter is changed at a time. The varied model parameters and best-fit magnetic susceptibility values are shown as insets.