Interpretation of the ultraviolet–visible spectraof malaria parasite Plasmodium falciparum
Yulia M. Serebrennikova,1,* Janus Patel,1 and Luis H. Garcia-Rubio2
1College of Public Health, University of South Florida, 13201 Bruce B. Downs Boulevard, Tampa, Florida 33830, USA2Claro Scientific LLC, 10100 Dr. Martin Luther King Jr. St. N., St. Petersburg, Florida 33706, USA
Spectrophotometric analysis of biological cells hasbeen a subject of continuous interest because it canprovide substantial information on the physical,chemical, and physiological character of the cells[1–3]. Such characterization of cells from simpleand nondestructive measurements has widespreadapplications in biology and medicine. In particular,the detection and identification of changes in cellsdue to diseases and/or external impact are of largesignificance. Yet, the majority of the studies have fo-cused exclusively on the scattering properties of thecells using backscattering, differential polarizedlight scattering, or angular scattering, which provideinformation on the size and shape of the cells but lit-tle on their chemical composition [4–6]. On the otherhand, exclusive focus on the extraction of the chemi-cal composition, such as nucleotides and proteins, ofthe cells by using Raman and/or IR spectroscopicmeasurements often lacks the information on thephysical structure of the cells [7,8].
Multiwavelength measurements with detectorshaving small acceptance angle (<2°) across the UV–visible spectroscopy portion of the electromagneticspectrum capture both attenuation of light due to ab-sorption and forward scattered light of the measuredcells [9]. Thus, the information on both physicalstructure and chemical composition of the cells canbe obtained from a single measurement [10]. Themeasured spectra can be interpreted with modelsbased on the appropriate choice of light scatteringtheory (Mie, Rayleigh–Debye–Gans, anomalous dif-fraction, etc.). This approach has been successfullytested for the interpretation of the UV–visible spec-troscopy spectra of various types of bacteria [10–12].
In this study, we extended these models to the ana-lysis of UV–visible spectroscopy spectra of an in-traerythrocytic parasite Plasmodium falciparum,which is responsible for highly severe and often fatalcases of malaria. During its growth within an ery-throcyte, a parasite digests most of the erythrocyte’shemoglobin and induces structural changes to thehost erythrocyte that lead to rupture [13]. Further,since the parasites lack the enzyme heme oxygenaserequired for completion of hemoglobin catabolism,the digested heme is mineralized into insoluble
and toxic hemozoin, which is sequestered in the di-gestive vacuole of the parasite [14]. Production of thistoxic pigment has been a target of many antimalarialdrugs [15]. Spectroscopic characterization can be aneffective tool to study the parasite’s biology, develop-ment, and reaction to the antimalarial treatment. Inparticular, accumulation of antimalarials in the di-gestive vacuole, a structural organelle where hemo-zoin is accumulated, and the production of hemozoincan be monitored.In this work we present a model based on Mie the-
ory and core-shell geometry for the interpretation ofthe spectral features of P. falciparum in terms of thecharacteristic constituents of the protozoan cells. Themodel was tested with the experimentally acquiredUV–visible spectroscopy spectra of different intracel-lular stages of P. falciparum extracted from the hosterythrocytes. In order to achieve accurate descriptionof the spectral features of the parasites, an estimateof the hemozoin refractive index was derived fromthe measured spectra of extracted pigment. We eval-uated the degree of reliability of the model by itscapability of reproducing the measured spectra, sen-sitivity analysis, and the ability to produce realisticestimates of the structural and compositional charac-teristics of the parasite cells.
2. Methods
A. Experimental
In vitro cultures of the W2 strain of P. falciparumwere grown in group Aþ erythrocytes at 4% hemato-crit in Royal Park Memorial Institute (RPMI)and 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES) media following the method describedin [16]. Parasitemia levels were estimated throughmicroscopic evaluation of Giemsa-stained thinsmears. The extraction of parasites was performedby lysis with saponin [13,17]. Briefly, 1ml of 0.1%saponin solution in deionized water or phosphatebuffered saline (PBS) was added to 0:5ml of approxi-mately 50% hematocrit erythrocyte suspension. Themixture was incubated at 37 °C with constantagitation for 15 min. Then, it was centrifuged at13; 000 rpm for 1 min and the pellet was resuspendedin PBS. The procedure was repeated 3–4 times to re-move the dissolved hemoglobin and the erythrocytemembrane pieces. Visible microscopy inspection wasperformed to confirm the integrity of the extractedparasites. Hemozoin was isolated through additionalhypo-osmotic lysis of parasites with deionized waterand mechanical breakdown by vortex.
The UV–visible spectroscopy spectra were re-corded using a diode array spectrometer (HP 8443Hewlett-Packard, Palo Alto, California) having an ac-ceptance angle smaller than 2°. All measurementswere conducted at room temperature using a 1 cmpath length cuvette. Prior to recording a sample spec-trum, the spectrometer was zeroed to account for anystray light. To avoid the effect of inhomogeneities inthe suspending medium, the background spectrumwas taken using the PBS batch utilized in the pre-paration of the protozoa samples or the deionizedwater used for the extraction of hemozoin.
B. Interpretation Model
The model used for the interpretation of the UV–visible spectroscopy spectra of P. falciparum cellswas based on Mie theory and on the recent develop-ments in the interpretation of spectral data [10–12].The advantages of using Mie theory are that it is notrestricted by particle size or the value of the refrac-tive index and it provides a robust solution to thescattering problem for volume equivalent spheres[18]. Although the shape of P. falciparum cells devi-ates from that of a sphere at the early stages of de-velopment, it approaches to spherical shape at themature stages. Further, sphere-equivalent approxi-mation for biological particles, such as bacteria, hasbeen successfully applied in the context of the UV–visible spectroscopy transmission measurements[10–12].
A model for the interpretation of the UV–visiblespectroscopy spectra of bacterial cells suggested by[10] approximates the bacterial turbidity spectrumas a weighted sum of contributions from bacterialmacrostructures (cell body) and internal structures,each modeled as a homogeneous Mie particle. Tomodel a protozoan cell, we used the same approxima-tion of additivity of the spectral contributions fromthe cell’s structural elements. The three mostprominent structural elements were identified forP. falciparum cells, namely, the digestive vacuole(DV), the nucleus (NU), and the organelles (ORG),as shown in Fig. 1. Each of these constituents wasassumed to be an independent scattering elementwithin the cell and, together with the cell body,constituted a structural group. In this study, the ex-tinction efficiency of each group was computedindependently using a core-shell sphere-equivalentmodel, where a nucleus, a digestive vacuole, or an or-ganelle was a core and the cell body was the shell.The core-shell geometry provides a better model tothe cell structure compared to a homogeneous
Fig. 1. Schematics of the P. falciparummodel: the protozoan cell is approximated as a weighted sum of three structural groups: cell bodyand digestive vacuole (DV), cell body and nucleus (NU), and cell body and structural organelles (ORG).
approximation, thus providing a natural infrastruc-ture for the introduction of the differences in the re-fractive indices and sizes of the cell components. Thetotal turbidity spectrum of a protozoan cell was mod-eled as the weighted sum of the contributions fromthe identified structural groups, as shown in Eq. (1):
τðλ0Þ ¼ Npℓ�π4
�fωDVQext;DVD2 þ ωNUQext;NUD2
þ ωORGQext;ORGD2g: ð1Þ
The parameterNp in Eq. (1) is the number density ofcells, ℓ is the path length, D is the sphere-equivalentdiameter of the cell, Qext corresponds to the extinc-tion efficiency, and λ0 is the wavelength. The massbalance constraint for the turbidity equation was im-plemented such that
ωDV þ ωNU þ ωORG ¼ 1: ð2Þ
The extinction efficiency for each structural groupwas computed as given by [18]:
Qext ¼2
x2X∞n¼1
ð2nþ 1ÞfReðan þ bnÞg; ð3Þ
where the size parameter x ¼ 2πn0D=λ0 is a ratio ofthe sphere-equivalent diameter of the cell to the in-cident wavelength. The series expansion coefficientsan and bn in Eq. (3) define the scattered radiationfield and are combinations of three Ricatti–Bessselfunctions and their derivatives. To achieve numericalstability of the algorithm for the computation of thean and bn coefficients for a core-shell sphere, theKerker’s equations were modified to replace all theRicatti–Bessel functions with their logarithmicderivatives and numerically stable ratios. The loga-rithmic derivatives and ratios were computed withdownward or upward recurrences as suggested by[19,20]. The coefficients an and bn are functions ofthe size parameters x (defined above) and x0 ¼2πn0D0=λ0, where D0 is the sphere-equivalent dia-meter of the core, and m and m0 are the complex re-fractive indices of the shell and the core, respectively.In the P. falciparummodel, x is the size parameter ofthe cell body and m denotes the refractive index ofcytoplasm, whereas x0 is the size parameter and m0
is the refractive index of the digestive vacuole, thenucleus, or the organelles.
The real n and imaginary k parts of the complexrefractive index of each structural component werefunctions of its chemical composition and were calcu-lated as weighted sums of the contributions fromma-jor compositional constituents [10]:
ki ¼PMi
j¼1 υijkj ni ¼PMi
j¼1 υijnj : ð4Þ
The mass balance of the fractions of compositionalconstituents for each structural component was im-plemented such that the sum of the fractions alwaysadds up to 1. The compositional constituents for eachstructural element considered in the model are givenin Table 1. Note that the chemical composition of theprotozoa cell components was somewhat simplified.In particular, the majority of the cell’s constituentsthat are nonabsorbing or weakly absorbing in thestudied wavelength region, which include proteins,polysaccharides, and lipids, were modeled with thesame average refractive index. This average re-fractive index was approximated with the Cauchyformula [21]:
nðλ0Þ ¼ a0 þa1
λ20þ a2
λ40þ…: ð5Þ
The first two parameters of Eq. (5) were used for re-presentation of the wavelength-dependent refractiveindex of the nonabsorbing macromolecules. It hasbeen shown that this approximation adequately re-presents the bulk optical properties of microorgan-isms [10,22]. The refractive indices of DNA, RNA,and nucleotides were estimated under assumptionof P. falciparum having guanine–cytosine content(%Gþ C) of 20% on average [23] and from the pre-viously obtained refractive indices of the purine andpyrimidine bases [10]. The refractive indices of waterand hemoglobin were obtained from the publisheddata [24,25].
The refractive index of hemozoin was estimatedfrom the measured spectra of the crystals extractedthrough lysis of mature stages of P. falciparum as fol-lows. An initial estimate of the imaginary part of thehemozoin refractive index was made from the mea-sured absorption spectrum. The corresponding realpart of the refractive index was estimated with theKramer-Kronig transforms [26]. Then, this initial
Table 1. Composition of the Structural Groups of the P. falciparum Interpretation Model
Structural Group (shell/core) Composition of the Shell Composition of the Core
estimate of the complex refractive index was used topredict a measured spectrum, and the measured andcalculated spectra were compared in the least-squaresense. The procedure was repeated with new esti-mates of the refractive index parameters until theminimal residual sum of squares of the fit wasachieved.To interpret each measured spectrum of P. falci-
parum, a theoretical spectrum was calculated, fol-lowing Eq. (1), as a function of selected structuraland compositional parameters of protozoa cells. Aleast-square iterative procedure was set up to fitthe measured and corresponding theoretical spectrain the 300–900nm wavelength range. The theoreti-cal spectrum was iterated as a function of the modelparameters until convergence was achieved (i.e., re-lative changes in the sum of squares were less than10−6). A Nelder–Meade downhill simplex optimiza-tion algorithm was used for this purpose [27]. Thevariable transformation techniques were used to im-plement the boundary constraints for the parameters[28]. The model parameters included the sphere-equivalent volumes of the whole cell, the digestivevacuole, and the nucleus, and the chemical composi-tion of each structural element, as given in Table 1.Notice that, although the number of parameters hasbeen increased relative to the homogeneous particleapproximation, the core-shell geometry results in acloser approximation to the scattering properties ofthe cells and the data contains sufficient informationfor their estimation (see Subsection 3.D).
3. Results
A. Estimation of the Optical Properties of Hemozoin
Several UV–visible spectroscopy spectra of hemozoinparticles extracted through the lysis of mature stagesof P. falciparum are shown in Fig. 2(a). The averagesphere-equivalent volume of the particles, estimatedwith the theoretical interpretation model, was 0:09�0:03 μm3, which is in agreement with the average vo-lume of 0:1 μm3 reported in [29,30]. The accuracy ofthe estimation can be appreciated with Fig. 2(a),where the measured spectra are compared with thecorresponding spectra predicted with the interpreta-tion model and the estimated refractive index of he-mozoin obtained as described above. The real andimaginary parts of the estimated hemozoin refrac-tive index are shown in Fig. 2(b).
B. Measured Spectra of the Extracted Malaria Parasites
Intraerythrocytic development of P. falciparumstarts with the early trophozoite stage, also knownas a ring stage, which is followed by the maturetrophozoite stage as the parasite grows, and is culmi-nated with the schizont stage. Considerable transfor-mation in the parasites’ physical structure andchemical composition occur during this development.These include changes in the size and shape of theprotozoa cells, internal morphology, and amountsof nucleotides and hemozoin [31]. Therefore, appreci-
able changes in the spectral features can be expected.The extraction experiments were designed to capturethe spectral features of the parasites at differentstages of development. Further, to capture the poten-tial variability in the spectral features at each stageand to ensure the quality of the extraction procedure,several independent experiments of the extraction ofparasites at each growth stage were conducted. Thereproducibility of the extraction procedure can be ap-preciated with Fig. 3, which compares six spectra ofthe P. falciparum ring stage obtained from three in-dependent extraction experiments. The reproducibil-ity of the measured spectra between the experimentsvalidates the extraction procedure.
The spectroscopic features of three developmentalstages are contrasted in Fig. 4(a). The correspondingfirst derivative spectra that enhance the differencesin the absorption spectral features are shown inFig. 4(b). Qualitative comparison of the measuredspectra and derivatives reveals considerable changein the spectral features of parasites with growth.
Fig. 2. (a) Comparison between the measured and predicted UV–visible spectroscopy spectra of hemozoin particles extracted frommature stages of P. falciparum. (b) The imaginary kðλÞ andreal nðλÞ parts of the estimated refractive index of the extractedhemozoin.
Two noticeable common features can be seen in thefirst derivative spectra of the parasites at all stages:the peak positioned around 650nm and the trough atapproximately 295nm; both are indicative of hemo-zoin [Fig. 4(b)]. The changes in these features withparasites’ development from the ring stage to theschizont stage indicate the increase in the intracellu-lar amount of hemozoin. The position of the Soretband in the measured spectra, at approximately412nm, was intermediate of those of hemozoin at408nm and oxyhemoglobin at 416nm. The presenceof hemoglobin in the parasites is also evident fromthe characteristic oxyhemoglobin doublet at 540 and575nm, which can be seen in the first derivatives ofthe spectra [Fig. 4(b)]. This doublet was more pro-nounced in the spectral features of the trophozoitestage. Finally, the peak at 263nm, which correspondsto the P. falciparum nucleic acid (Gþ C content of20%), increased from the ring to the schizont stage,indicating buildup of nucleotide material [Fig. 4(b)].
C. Interpretation of the Measured Spectra of the ExtractedMalaria Parasites
The approximation of the morphological structure ofP. falciparum cells was based on the informationavailable from the published literature. The para-site’s cell was approximated to consist of the cell bodyfilled with cytoplasm, digestive vacuole, nucleus, andorganelles. The chemical composition of each struc-tural component (Table 1) was modeled to accountfor the major chromophoric groups of the parasitesand refractivity of the nonabsorbing compounds. Thefeatures recognized from the qualitative examina-tion of the measured spectra were also considered forthe construction of the interpretation model.
The results of the interpretation of the spectra ofP. falciparum at three developmental stages areshown in Figs. 5–7. Each plot contrasts a measuredspectrum of the extracted P. falciparum cells and thecorresponding spectrum predicted with the interpre-tation model. In addition, the spectral contributionsof the model components (digestive vacuole, nucleus,and organelles) to the total predicted spectrum areshown. The residuals of the fits between the mea-sured and calculated spectra are also included in thefigures.
The examples of the spectral interpretation illu-strated with Figs. 5–7 indicated the reliability ofthe interpretation approach, as the model performedwell for the analysis of each spectrum of differentdevelopmental stages. The low residuals of the fit be-tween the measured and predicted spectra indicatedthat the interpretation model adequately replicatedthe measured spectral features of the protozoa. The
Fig. 3. UV–visible spectroscopy spectra of P. falciparum ringstages extracted from three independently prepared erythrocytecultures (the spectra of one to three replicate samples extractedfrom each culture are shown). The optical density spectra werenormalized with their corresponding 230–900nm averages toeliminate the effect of cell number density and, thus, to allow di-rect comparison of the spectral features.
Fig. 4. (a) Comparison of the UV–visible spectroscopy spectra ofP. falciparum ring, trophozoite, and schizont stages extracted fromerythrocyte cultures. The optical density spectra were normalizedwith their corresponding 230–900nm averages to eliminate the ef-fect of cell number density and, thus, to allow direct comparisonof the spectral features. (b) Comparison of the corresponding firstderivatives.
spectral contributions of each structural componentto the total spectrum were significant for all develop-mental stages, justifying the complex structure of theinterpretation model. The values of the model para-meters estimated from the measured spectra aresummarized in Table 2. There was good agreementbetween the results of the interpretation analysisof the independently measured spectra for eachstage. The variance in the estimated parametervalues was overall less than 50% for six indepen-dently measured spectra of each stage. This variancein the estimated parameter values included the
measurement and analysis errors as well as the var-iance due to natural variability among the indepen-dent samples. Given the low residuals of the fitsbetween the measured and predicted spectra, thenatural variability was the most likely the cause ofthe variance in the estimated parameter values. Thisis supported by the fact that the estimates of thestructural parameters, such as total cell volumeand the volumes of the digestive vacuole, the orga-nelles and the nucleus, were consistent among theindependent samples, whereas the chemical compo-sition parameters, such as the amounts of hemozoin,hemoglobin, and nucleotides per cell, showed largervariability.
D. Sensitivity Analysis of the Interpretation Model
To evaluate the structure of the interpretation mod-el, sensitivity analysis was conducted. In particular,the correlation between the parameters and effect ofthe variability in the parameter values on the totalpredicted spectrum were assessed. For this purpose,two theoretical spectra were generated using theaverage values of the parameters estimated for theP. falciparum ring and trophozoite stages (Table 2).Then, the value of each model parameter was per-turbed by �50% for each test case and the changesin the calculated spectral intensities were averagedfor all wavelengths. The results, summarized inTable 3, show that, in the case of the ring stage,the model was the most sensitive to the structuralparameters, such as the mean cell volume (MCV), vo-lumes of digestive vacuole (DVV) and nucleus (NUV),and fraction of organelles (FOC), and to composi-tional parameters, such as the amount of hemozoin(HZ) and the concentration of proteins in cytoplasm(FPC). Smaller sensitivity to the rest of the model
Fig. 5. Comparison between the measured UV–visible spectro-scopy spectrum (τmeas) and the corresponding predicted spectrum(τcalc) of the P. falciparum ring stage extracted from erythrocytecell culture. The difference between the measured and predictedspectra (σres) and the predicted spectral contribution from the nu-cleus (τNU), organelles (τORG), and digestive vacuole (τDV) structur-al components are shown.
Fig. 6. Comparison between the measured UV–visible spectro-scopy spectrum (τmeas) and the corresponding predicted spectrum(τcalc) of the P. falciparum trophozoite stage extracted from ery-throcyte cell culture. The difference among the measured and pre-dicted spectra (σres) and the predicted spectral contribution fromthe nucleus (τNU), organelles (τORG), and digestive vacuole (τDV)structural components are shown.
Fig. 7. Comparison between the measured UV–visible spectro-scopy spectrum (τmeas) and the corresponding predicted spectrum(τcalc) of the P. falciparum schizont stage extracted from erythro-cyte cell culture. The difference between the measured and pre-dicted spectra (σres) and the predicted spectral contributionfrom the nucleus (τNU), organelles (τORG), and digestive vacuole(τDV) structural components are shown.
parameters was due to the smaller values of thoseparameters found for the P. falciparum ring stageand, therefore, to smaller influence on the total pre-dicted spectrum. Yet, the sensitivity increased forsuch parameters as the fraction of proteins in the nu-cleus (FPN) and mean organelle volume (MOV) atthe trophozoite stage (Table 3). Further, althoughthe model sensitivities to the concentrations ofhemoglobin (HB) and nucleotides (TNN and TNC)appeared to be small when the total wavelengthrange was considered (Table 3), they were fivefold totenfold greater within the 250–450nm wavelengthrange, where the contributions of these parameterswere the greatest. These results indicated that allmodel parameters contributed to the prediction ofthe measured spectra.
4. Discussion
The statistical evaluation of the interpretation ana-lysis with the residuals of the fit between the mea-sured and predicted spectra and the sensitivity tothe model parameters indicated the adequacy ofthe prediction and the robustness of the model struc-ture. The next step was to validate the structural andcompositional characteristics of the P. falciparumcells estimated with the interpretation model. Forthis purpose, the model outcomes were compared tothe available published data for the P. falciparumstructure and composition. A summary of such infor-mation obtained from published reports and electronmicrographs is compiled in Table 4.The volume of P. falciparum cell changes from
5–10 μm3 at the ring stage to 30� 10 μm3 at the ma-ture trophozoite stage [32]. It can further increase upto 50� 10 μm3 during the schizont stage [32,33]. Theestimates of cell volume obtained from the interpre-tation analysis of the measured spectra (Table 2)
were in very good agreement with the published datafor the ring and trophozoite stages and were slightlyless than expected for the schizont stage. Yet, the sizeparameter estimates were in agreement with the es-timates made from microscopic examination, whichrevealed that the parasites extracted from the ery-throcytes were smaller in size that those withinthe erythrocytes. This feature was attributed to thetreatment of the cells with saponin.
The digestive vacuole of the parasite is known to bealready formed at the early stages. Its volume hasbeen approximated to be 4 μm3 [34]; however it canreach 2:5–2:8 μm in diameter [33], which correspondsto a sphere-equivalent volume of 9–11 μm3 at latedevelopmental stages. The model’s estimate of theparasite’s vacuole was approximately 3 μm3 forthe ring stage, which is in good agreement with thepublished data. The twofold larger estimates of thedigestive vacuole volume for the trophozoite stageare in general agreement with [33]. The estimatedvacuole volumes for the schizont stage were some-what smaller compared to the estimates for the tro-phozoite stage. A reasonable explanation can be thefact that the parasite’s vacuole becomes compactedinto a dense rounded mass late in the schizontstage [33].
The examination of the published electron micro-graphs suggested that the nucleus occupies 10%–
25% of the total cell volume [31]. The model’s resultswere in agreement with this estimate: the volumefractions of the nuclei were estimated to be 11.3%,17.7%, and 26% on average for the ring, trophozoite,and schizont stages, respectively. The increase in thesize of nucleus with the parasite’s development is ex-pected, since the nucleic acid material is multipliedand the nucleus enlarges and divides into 16–32nuclei by the late schizont stage [31,33]. Similar
Table 2. Summary of the Values of the Structural and Compositional Parametersof P. falciparum Stages Obtained with the Interpretation Model
Table 3. Sensitivity of the Model Computed as the Percent Change in the Calculated Optical Densitya in responseto the �50% Perturbation of the Model Parameter Values of the Ring and Trophozoite Stages
changes were seen in the estimated amount of nu-cleotides (Table 2). Since P. falciparum genome sizeis 13–23 million base pairs [23], the minimal ex-pected amount of nucleotides per cell is 5:5–9:8 fg. Gi-ven that nearly equivalent amounts of RNA andother nucleotides should be present in nucleus,and twofold to fivefold greater amounts of nucleo-tides can be expected in the rest of the cell’s body, themodel’s estimates of nucleotide concentrations forthe ring stage are very reasonable. With furtherdevelopment, active synthesis of RNA and the begin-ning of DNA replication should increase the amountof nucleotides per cell at the trophozoite stage [31].The interpretation model’s estimates were in agree-ment: there was a fivefold to sixfold increase in theamount of nucleotides in nucleus and a twofold in-crease in the nucleotide amount in the cell body(Table 2). Continuing replication of DNA duringthe schizont stage should result in an increase of16–32 times in the amount of DNA compared tothe ring stage [31,33]. Because of the active growthprocess at the schizont stage, larger amounts of RNAshould also be expected (Table 4). The model’s esti-mates were in agreement with the expected values(Table 2). Further, active nucleotide synthesis shouldaccount for the larger amounts of proteins estimatedfor the late developmental stages compared to thering stage (Table 2). The estimated amount of orga-nelles was overall realistic for the eukaryotic cells.The estimated mean organelle size increased withthe development from ring to trophozoite as mostof the structural organelles, such as the endoplasmicreticulum and the mitochondrion, enlarge by the tro-phozoite stage [31].The estimated amount of hemozoin, the most char-
acteristic feature of the Plasmodium spp. also chan-ged between the ring and the trophozoite stages,and between the trophozoite and schizont stages (Ta-ble 2). It was the lowest, approximately 0:22 pg=cell,for the ring stage, which was in agreement with thereported estimates of 0:15–0:45 pg=cell [34]. Themodel’s estimates for the amount of hemozoin atthe trophozoite stage were also in excellent agree-ment with the published experimental data of0:52� 0:05pg=cell, according to [13]. Since parasitescontinue to ingest hemoglobin until quite late in theschizont stage [33], hemozoin concentration canreach 1:0� 0:1pg=cell, assuming that up to 90% ofthe erythrocyte’s hemoglobin is converted to hemo-
zoin at this stage [35]. The amount of hemozoinestimated with the model followed the expected pat-tern and increased from the trophozoite stage tothe schizont stage. Finally, small amounts of nondi-gested hemoglobin were estimated for the tropho-zoite and schizont stages (Table 2); it has beenreported that approximately 4%� 4% of iron con-tained in the digestive vacuole is still in the formof hemoglobin at the trophozoite stage [13].
5. Conclusion
An interpretation model based on core-shell Miescattering calculations has been applied to analy-sis of the UV–visible spectroscopy spectra of threedevelopmental stages of the malaria parasiteP. falciparum. The model was formulated to accountfor the size and composition of the cell body and thoseof three populations of organelles (digestive vacuole,nucleus, and average structural organelle). The mod-el was equally accurate in predicting the spectral fea-tures of the parasites at different developmentalstages. The results showed that all three populationsof organelles considered by the model play signifi-cant roles in absorption and scattering of light byP. falciparum cells. The model’s estimates of thestructural and compositional parameters of the cellswere in very good agreement with published dataand/or estimates obtained from published electronmicrographs of P. falciparum. The spectral featuresof P. falciparum were found to be considerably differ-ent among the developmental stages. The results ofthe interpretation analysis indicated that it is possi-ble to quantitatively track the changes in the cellularstructure and the concentrations of the most promi-nent chemical constituents of the parasite cells. Inparticular, the ability to follow the changes in theamounts of hemozoin and nucleotides would haveimportant applications in the research on malariaantibiotic resistance and control.
This work was supported by a Florida High TechCorridor 09-08 Industry Seed grant (6408-1024-00).The authors are grateful to Dennis Kyle and WilburMilhous at the College of Public Health, Universityof South Florida, for assistance with the preparationof P. falciparum cultures, and to Debra Huffman atClaro Scientific LLC (St. Petersburg, Florida) for as-sistance with microscopic observations.
Table 4. Summary of the Estimates of Structural and Compositional Parametersof P. falciparum Obtained from the Published Literature
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