Characterization of red blood cells with multiwavelength transmission spectroscopy.

Multiwavelength transmission (MWT) spectroscopy was applied to the investigation of the morphological parameters and composition of red blood cells (RBCs). The MWT spectra were quantitatively analyzed with a Mie theory based interpretation model modified to incorporate the effects of the nonsphericity and orientation of RBCs. The MWT spectra of the healthy and anemic samples were investigated for the RBC indices in open and blinded studies. When MWT performance was evaluated against a standard reference system, very good agreement between two methods, with R (2) > 0.85 for all indices studied, was demonstrated. The RBC morphological parameters were used to characterize three types of anemia and to draw an association between RBC morphology and anemia severity. The MWT spectra of RBCs infected with malaria parasite Plasmodium falciparum at different life cycle stages were analyzed for RBC morphological parameters. The changes in the RBC volume, surface area, aspect ratio, and hemoglobin composition were used to trace the morphological and compositional alterations in the infected RBCs occurring with parasites' development and to provide insights into parasite-host interactions. The MWT method was shown to be reliable for determination of the RBC morphological parameters and to be valuable for identification of the RBC pathologic changes and disease states.

Spectrophotometric detection of susceptibility to anti-malarial drugs.

BACKGROUND: With malaria drug resistance increasing in prevalence and severity, new technologies are needed to aid and improve the accuracy and clinical relevance of laboratory or field testing for malaria drug resistance. This study presents a method based on simple and reagentless spectroscopic measurements coupled with comprehensive spectral interpretation analysis that provides valuable quantitative information on the morphological and compositional responses of Plasmodium falciparum and infected red blood cells (IRBCs) to anti-malarial treatment. METHODS: The changes in the size, internal structure, nucleotide and haemozoin composition of the parasites as well as the morphology (size and shape) and haemoglobin composition of the IRBCs treated with dihydroartemisinin (DHA) and mefloquine (MFQ) were investigated using a spectral interpretation analysis. RESULTS: DHA treatment reduced the sizes of the parasites and their structural organelles. The haemoglobin composition of the host IRBCs determined from spectroscopic analysis changed negligibly following DHA treatment. MFQ treated parasites grew to the same size as those from parallel non-treated cultures but lacked haemozoin. Lesser deformation of the cell shape and no haemoglobin depletion were detected for the IRBCs of MFQ treated cultures. CONCLUSIONS: The spectroscopic analysis method proved to be sensitive for recognition of the effects of anti-malarial treatment on the structure and composition of the parasites and IRBCs. The method can have significant potential for research and clinical applications such as evaluating patient specimens for drug action, drug effects or for therapeutic monitoring.

Multiwavelength transmission spectroscopy revisited for the characterization of the protein and polystyrene nanoparticle mixtures.

Multiwavelength Transmission (MWT) UV-Vis-NIR spectroscopy, an effective technique often underutilized for the characterization of processes involving particulates, such as protein aggregation, is systematically explored using bovine serum albumin and a set of NIST-traceable particle size (PS) standards having certified particle diameters over the nominal size range of 30 to 100 nm. The PS standards are used as surrogates for protein aggregates and other contaminants such as oils and microbubbles. Therefore, the standards can be used to quantitatively modify the optical properties of protein solutions and thus observe the effect of the presence of aggregates and other particulates on their wavelength-dependent transmission spectra. The experimental results demonstrate that the changes induced in the optical density spectra of proteins due to the presence of PS particles are detectable and consistent with the expectations set by light scattering theory. It is demonstrated that the size and relative concentrations of the particle populations present in the protein samples can be quantified. Because of the considerable dynamic range of MWT UV-Vis-NIR spectroscopy for particle analysis and its real-time measurement capabilities, this type of spectroscopy can be effectively used for the characterization of protein aggregates and for the continuous real-time monitoring of aggregation processes and for the identification and quantification of contaminants in protein-based products.

Multiwavelength transmission spectroscopy revisited for the characterization of the protein and polystyrene nanoparticle interactions.

Multiwavelength transmission ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy is an effective technique that has not yet been fully exploited for the characterization of products of protein and particle interactions. Here, it is explored by using bovine serum albumin and National Institute of Standards and Technology-traceable particle size standard having a nominal diameter of 20 nm. Adsorption of bovine serum albumin to the particles is quantitatively ascertained through its effect on the wavelength-dependent transmission spectra of protein and particle mixtures. The experimental results demonstrate that the changes induced in the transmission spectra of protein and particle mixtures because of protein adsorption on particles are detectable and consistent with the expectations set by the light-scattering theory. The size, structure, composition, and relative concentrations of the particle populations present in the protein-particle mixtures can be quantified. Given the considerable dynamic range of multiwavelength transmission UV-Vis-NIR spectroscopy for particle analysis and its real-time measurement capabilities, this type of spectroscopy can be effectively used for the characterization of the products of protein-particle interaction and for the continuous real-time monitoring of interaction processes.

Multi-wavelength transmission spectroscopy revisited for micron and submicron particle characterization.

Multi-wavelength transmission (MWT) ultraviolet-visible-near-infrared (UV-Vis-NIR) spectroscopy, a technique underappreciated for particle characterization, is systematically explored using a set of NIST traceable standards over the nominal size range of 20 to 20,000 nm. Experimental results demonstrate that the particle size distributions obtained from MWT spectral data are in excellent agreement with the values reported by the manufacturer. In addition, it is shown that quantitative information on the particle concentration can be obtained--which is not currently accessible from commercially available light scattering instrumentation. The results validate that MWT UV-Vis-NIR spectroscopy has a considerable dynamic range for particle size measurements and offers significant advantages over other particle characterization techniques. Among these are the simplicity of the instrumentation and the measurements and the wealth of quantitative information contained in the MWT spectra. Most importantly, with standardized measurement protocols and standardized spectrometer configurations, MWT measurements can be used to provide the user and the manufacturer of particles with traceable data (i.e., the spectra and the quantitative analysis) for quality assurance.

Hypochromicity in red blood cells: an experimental and theoretical investigation.

Multiwavelength UV-visible transmission spectrophotometry is a useful tool for the examination of micron-size particle suspensions in the context of particle size and chemical composition. This paper reports the reliability of this method to characterize the spectra of purified red blood cells both in their physiological state and with modified hemoglobin content. Previous studies have suggested the contribution of hypochromism on the particle spectra caused by the close electronic interaction of the encapsulated chromophores. Our research shows, however, that this perceived hypochromism can be accounted for by considering two important issues: the acceptance angle of the instrument and the combined scattering and absorption effect of light on the particles. In order to establish these ideas, spectral analysis was performed on purified and modified red cells where the latter was accomplished with a modified hypotonic shock protocol that altered the hemoglobin concentration within the cells. Moreover, the Mie theory was used to successfully simulate the spectral features and trends of the red cells. With this combination of experimental and theoretical exploration, definition of hypochromism has been extended to two subcategories.

Modeling and interpretation of extinction spectra of oriented nonspherical composite particles: application to biological cells.

The majority of cells and microorganisms have a nonspherical shape and complex structure that challenge the interpretation of their spectral features. To address this issue, two approximations to the core-shell Mie theory were proposed. These included the approximation of light extinction by an ellipsoid with representation of the extinction by an equivalent sphere and representation of the extinction by a population of ellipsoidal particles with those of two weighted particle orientations. These hypotheses were first tested through numerical interpretation of the theoretical extinction spectra of prolate nucleated ellipsoids mimicking biological cells generated with anomalous diffraction approximation used as a reference method. Theoretical cases of fixed and random particle orientations demonstrated excellent capabilities of the proposed approach to retrieve the size, shape, and composition parameters of the model particles. Second, the UV-visible spectra of Leishmania species, promastigotes, elongated cells with prominent nuclei, were interpreted. The retrieved estimates of the protozoa size, shape, nucleus size, and nucleotide composition were in agreement with the corresponding microscopy estimates and literature values. Both theoretical tests and experimental results illustrated that the proposed approach can be successfully applied to estimate the structural and compositional parameters of cells from spectroscopic measurements.

Quantitative analysis of morphological alterations in Plasmodium falciparum infected red blood cells through theoretical interpretation of spectral measurements.

Spectroscopic analysis can provide valuable insights into morphological and biochemical cellular transformations caused by diseases. However, traditional spectroscopic methods and the corresponding spectral interpretation approaches have been challenged by the complexities of the cell shape, orientation, and internal structure. Here we present an elegant spectral interpretation model that enables accurate quantitative analysis of the UV-visible spectra of red blood cells (RBCs) parasitized by the lethal human malaria parasite, Plasmodium falciparum. The model is based on the modified Mie theory (MMT) approach that incorporates the effects of the nonsphericity and orientation and multilayered cell structure to account for complex composition of the infected RBCs (IRBCs). We determine the structure and composition of the IRBCs and address unresolved matters over the alterations induced by the intraerythrocytic development of P. falciparum. The results indicate deformation and swelling of the IRBCs during the trophozoite stage of P. falciparum that is followed by substantial shrinkage during the schizont stages. We determine that up to 90% depletion of hemoglobin from the RBC cytosol does not lead to a net loss of iron from the infected cells. We quantitatively follow the morphological changes in the parasites during the intraerythrocytic development by applying the interpretation model to the UV-visible spectroscopic measurements of the IRBCs. We expect this method of quantitative spectroscopic characterization of the diseased cells to have practical clinical utility for rapid diagnosis, therapeutic monitoring, and drug susceptibility testing.

Interpretation of the ultraviolet-visible spectra of malaria parasite Plasmodium falciparum.

The absorption and scattering properties of three developmental stages of protozoan parasite Plasmodium falciparum were studied both experimentally and theoretically. Experimentally, the light attenuation and forward scattering from parasites extracted from host erythrocyte cultures were measured with UV-visible spectroscopy. The measured spectra were interpreted theoretically with a model based on the core-shell Mie theory in terms of the structural and compositional characteristics of the protozoa. The model accurately reproduced the features of the measured spectra of all developmental stages. The results show that realistic quantitative estimates of the parasite size, nucleotide, and hemozoin contents can be derived from the UV-visible spectroscopy measurements.

New method for the detection of micro-organisms in blood: application of quantitative interpretation model to aerobic blood cultures.

The physical and chemical changes occurring in blood that has been inoculated into a blood culture bottle can be used as means to detect the presence of microorganisms in blood cultures. These changes include primarily the conversion of oxy- to deoxyhemoglobin within the red blood cells (RBCs) and changes in the cell number densities. These changes in the physical and chemical properties of blood can be readily detected using spectrophometric methods thus enabling the continuous monitoring of blood culture vials to provide quantitative information on the growth behavior of the microorganisms present. This paper reports on the application of spectrophotometric information obtained from diffuse reflectance measurements of aerobic blood cultures to detect microbial growth and compares the results to those obtained using the standard blood culture system.

Quantitative interpretations of Visible-NIR reflectance spectra of blood.

This paper illustrates the implementation of a new theoretical model for rapid quantitative analysis of the Vis-NIR diffuse reflectance spectra of blood cultures. This new model is based on the photon diffusion theory and Mie scattering theory that have been formulated to account for multiple scattering populations and absorptive components. This study stresses the significance of the thorough solution of the scattering and absorption problem in order to accurately resolve for optically relevant parameters of blood culture components. With advantages of being calibration-free and computationally fast, the new model has two basic requirements. First, wavelength-dependent refractive indices of the basic chemical constituents of blood culture components are needed. Second, multi-wavelength measurements or at least the measurements of characteristic wavelengths equal to the degrees of freedom, i.e. number of optically relevant parameters, of blood culture system are required. The blood culture analysis model was tested with a large number of diffuse reflectance spectra of blood culture samples characterized by an extensive range of the relevant parameters.

Rayleigh-Debye-Gans as a model for continuous monitoring of biological particles: Part II, development of a hybrid model.

Rayleigh-Debye-Gans and Mie theory were previously shown to disagree for spherical particles under ideal conditions4. A Hybrid model for spheres was developed by the authors combining Mie theory and Rayleigh- Debye-Gans. The hybrid model was tested against Mie and Rayleigh- Debye-Gans for different refractive indices and diameter sizes across the UV-Vis spectrum. The results of this study show that the hybrid model represents a considerable improvement over Rayleigh-Debye-Gans for submicron particles and is computationally more effective compared to Mie model. The development of the spherical hybrid model establishes a platform for the analysis of non-spherical particles.

Rayleigh-Debye-Gans as a model for continuous monitoring of biological particles: Part I, assessment of theoretical limits and approximations.

A rapid tool for the characterization of submicron particles is light spectroscopy. Rayleigh-Debye-Gans and Mie theories provide light scattering solutions that can be evaluated within the time constants required for continuous real time monitoring applications, as in characterization of biological particles. A multiwavelength assessment of Rayleigh-Debye-Gans theory for spheres was conducted over the UV-Vis wavelength range where strict adherence to the limits of the theory at a single wavelength could not be met. Reported corrections to the refractive indices were developed to extend the range of application of the Rayleigh-Debye-Gans approximation. The results of this study show that there is considerable disagreement between Rayleigh-Debye-Gans and Mie theory across the UV-Vis spectrum.

Quantitative spectroscopy analysis of prokaryotic cells: vegetative cells and spores.

Multiwavelength ultraviolet/visible (UV-Vis) spectra of microorganisms and cell suspensions contain quantitative information on properties such as number, size, shape, chemical composition, and internal structure of the suspended particles. These properties are essential for the identification and classification of microorganisms and cells. The complexity of microorganisms in terms of their chemical composition and internal structure make the interpretation of their spectral signature a difficult task. In this paper, a model is proposed for the quantitative interpretation of spectral patterns resulting from transmission measurements of prokaryotic microorganism suspensions. It is also demonstrated that different organisms give rise to spectral differences that may be used for their identification and classification. The proposed interpretation model is based on light scattering theory, spectral deconvolution techniques, and on the approximation of the frequency dependent optical properties of the basic constituents of living organisms. The quantitative deconvolution in terms of the interpretation model yields critical information necessary for the detection and identification of microorganisms, such as size, dry mass, dipicolinic acid concentration, nucleotide concentration, and an average representation of the internal scattering elements of the organisms. E. coli, P. agglomerans, B. subtilis spores, and vegetative cells and spores of Bacillus globigii are used as case studies. It is concluded that spectroscopy techniques coupled with effective interpretation models are applicable to a wide range of cell types found in diverse environments.

Growth behavior of microorganisms using UV-Vis spectroscopy: Escherichia coli.

Multiwavelength transmission spectra of microorganisms and cell suspensions consist of combined absorption and scattering phenomena resulting from the interaction of light with microorganisms or cells typically suspended in a nonabsorbing media. The distribution of intensities as a function of wavelength depends on the size, shape, and optical properties of the sample. The optical properties are functions of the chemical composition and the state of aggregation, or association, of the chromophoric groups contained in the microorganisms. This article explores the growth behavior of Escherichia coli from the perspective of multiwavelength UV-Vis spectroscopy. Experimentally, it is demonstrated that the spectral signatures of the microorganism evolve as a function of time. It is also demonstrated that the spectral changes observed during growth are consistent with data reported elsewhere. From the theoretical point of view, it is demonstrated that the spectral signatures can be adequately represented with an interpretation model based on light-scattering theory. The parameters from the interpretation model reflect changes in size and chemical composition known to take place in the microorganisms during growth.

UV-visible spectrophotometric approach to blood typing II: phenotyping of subtype A2 and weak D and whole blood analysis.

BACKGROUND: A recently introduced quantitative blood typing approach uses antibody-induced changes in the UV-visible spectra of blood. Changes in the blood spectra's slope, caused by RBC agglutination, are translated into a numerical agglutination index (AI). Comparing the AI value against an established threshold yields a "yes and/or no" output from which to determine the phenotype. The efficacy and flexibility of this approach with whole blood use and the ability to analyze weak D, A2, and A2B were examined. STUDY DESIGN AND METHODS: Two hundred randomly selected blood bank donor samples were coded and forward typed directly from whole blood by using the spectrophotometric analysis. Reverse grouping on plasma from each sample was carried out with a new modified procedure by using higher ratios of plasma to RBCs. Results were compared to typing by an FDA-cleared automated typing system. Twenty-seven weak D samples, 15 A2 and 12 A2B, were similarly analyzed from whole blood. PEG improved detection of weak D, A2 and A2B subtypes. RESULTS: All two hundred coded samples were accurately typed, yielding identical results to the blood bank analysis in both forward and reverse grouping. All the weak D samples and A2 and A2B samples were clearly identified, having AIs above the type threshold indicator value. CONCLUSION: Spectrophotometric blood typing successfully phenotyped ABO and D in 200 whole blood samples. Reverse grouping of plasma was equally successful. The same method can identify weak D and A2 and A2B subtypes.