Research Fellow, Monash University
Raman and Infrared spectroscopy based applications for clinical research and diagnosis
My research focuses on the development of solutions to clinical problems based on vibrational spectroscopy, including point-of-care, nano-imaging and chemometrics applications. One of our topics is the diagnosis of malaria based on ATR-FTIR spectroscopy. Malaria, caused by Plasmodium falciparum, is one of the deadliest diseases, resulting in up to 1.2 million fatalities per annum. Accurate and early diagnosis followed by immediate treatment of the infection is essential to reduce mortality and prevent overuse of antimalarial drug. We have demonstrated that ATR-FTIR provides a low-cost, reagent-free and point-of-care tool for malaria diagnosis. Minimal invasiveness of this approach combined with versatility of the technique, its low cost, simplicity and high speed fit perfectly the requirements for developing countries. The plasmodium parasite provokes significative changes in the phenotype of the Red blood cell (e.g. DNA, lipids and Hemozoin), and this changes are detected by the IR spectrum, which contains a snapshot of the composition of the blood components. For that, we perform multivariate statistical analysis such as PLSDA or SVM on a dataset containing both, infected and control samples. We are currently performing field trials in Papua-New-Guinea and Thailand. Also, we studied malaria parasites and the effects of antimalarial drugs on them at the nanoscale level using AFM-IR and Raman and infrared microspectroscopy.
Abstract: Attenuated total reflectance mid-infrared spectra of serum and blood samples were obtained from 4,000 to 600 cm(-1). Models for the determination of albumin, immunoglobulin, total globulin, and albumin/globulin coefficients were established for serum samples, using reference data obtained by capillary electrophoresis. Based on the use of the amide bands I and II regions, the relative root mean square error of prediction (RRMSEP) was 4.9, 14.9, 4.5, and 7.1% for albumin, immunoglobulin, total globulin, and albumin/globulin coefficients, respectively, determined in an independent validation set of 120 samples using 200 samples for calibration. Additionally, the use of Kennard-Stone method for the selection of a representative calibration subset of samples provided comparable results using only 60 samples. For whole blood analysis, hemoglobin was determined in 40 validation samples using models built from 40 calibration independent samples with RRMSEP of 8.3, 5.5, and 4.9% with models built from direct spectra in the first case and from sample spectra recorded after lysis by sodium dodecyl sulfate and freezing, respectively, for the last two ones. The developed methodologies offer green alternatives for patient diagnosis in a few minutes, minimizing the use of reagents and residues and being adaptable for its use as a point-of-care method.
Pub.: 02 May '12, Pinned: 25 Aug '17
Abstract: Locally weighted partial least squares regression (LW-PLSR) has been applied to the determination of four clinical parameters in human serum samples (total protein, triglyceride, glucose and urea contents) by Fourier transform infrared (FTIR) spectroscopy. Classical LW-PLSR models were constructed using different spectral regions. For the selection of parameters by LW-PLSR modeling, a multi-parametric study was carried out employing the minimum root-mean square error of cross validation (RMSCV) as objective function. In order to overcome the effect of strong matrix interferences on the predictive accuracy of LW-PLSR models, this work focuses on sample selection. Accordingly, a novel strategy for the development of local models is proposed. It was based on the use of: (i) principal component analysis (PCA) performed on an analyte specific spectral region for identifying most similar sample spectra and (ii) partial least squares regression (PLSR) constructed using the whole spectrum. Results found by using this strategy were compared to those provided by PLSR using the same spectral intervals as for LW-PLSR. Prediction errors found by both, classical and modified LW-PLSR improved those obtained by PLSR. Hence, both proposed approaches were useful for the determination of analytes present in a complex matrix as in the case of human serum samples.
Pub.: 20 Apr '13, Pinned: 25 Aug '17
Abstract: Selected volatile organic compounds (VOCs) in breath may be considered biomarkers if they are indicative of distinct diseases or disease states. Given the inherent molecular selectivity of vibrational spectroscopy, infrared sensing technologies appear ideally suitable for the determination of endogenous VOCs in breath. The aim of this study was to determine that mid-infrared (MIR; 3-20 µm) gas phase sensing is capable of determining isoprene in exhaled breath as an exemplary medically relevant VOC by hyphenating novel substrate-integrated hollow waveguides (iHWG) with a likewise miniaturized preconcentration system. A compact preconcentrator column for sampling isoprene from exhaled breath was coupled to an iHWG serving simultaneously as highly miniaturized gas cell and light conduit in combination with a compact Fourier transform infrared spectrometer. A gas mixing system enabled extensive system calibration using isoprene standards. After system optimization, a calibration function obtaining a limit of quantification of 106 ppb was achieved. According to the literature, the obtained sensitivity is sufficient for quantifying middle to high isoprene concentrations occurring in exhaled breath. Finally, a volunteer breath sample was analysed proving comparable values of isoprene in a real-world scenario. Despite its fundamental utility, the proposed methodology contains some limitations in terms of sensitivity and temporal resolution in comparison with the readily available measurement techniques that should be addressed during future optimization of the system. Nonetheless, this study presents the first determination of endogenous VOCs in breath via advanced hollow waveguide MIR sensor technology, clearly demonstrating its potential for the analysis of volatile biomarkers in exhaled breath.
Pub.: 23 May '14, Pinned: 25 Aug '17
Abstract: In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measurements. By using malaria diagnosis as an example, we demonstrate that combining the non-fundamental and fundamental regions of the RR spectrum improves the sensitivity and diagnostic capability of the technique. The discovery will have considerable implications for the ongoing development of Raman spectroscopy for blood disease diagnoses and monitoring heme perturbation in response to environmental stimuli.
Pub.: 27 Sep '14, Pinned: 25 Aug '17
Abstract: The widespread and cost-effective use of transflection substrates in Fourier transform infrared (FTIR) imaging of clinical samples is affected by the presence of artefacts including the electric field standing wave (EFSW) and contributions from light dispersion. For IR-based diagnostics, the manifestation of undesirable artifacts can distort the spectra and lead to erroneous diagnosis. Nevertheless, there is no clear consensus in the literature about the degree of influence of these effects. The aim of this work is to contribute to this discussion by comparing transflection and transmission images of the same tissue. For this purpose two adjacent sections of the same tissue (lymphoma sample) were fixed onto a CaF2 window and a transflective slide for FTIR imaging. The samples in this case had a central area where based on morphology it was presumed the fixative did not penetrate to the same extent hence providing a comparable region for the two different substrates with a distinct physical/chemical difference. Transmission and transflection spectra from adjacent hyperspectral tissue images were combined in an extended dataset. Surprisingly, unsupervised hierarchical cluster analysis clustered together transflection and transmission spectra, being classified according to differences in tissue fixation instead of the geometry employed for the image acquisition. A more detailed examination of spectra from the peripheral zone of the tissue indicated that the main differences between the transflection and transmission spectra were: (1) a small shift of the amide I, (2) a larger "noise" component in the transflection spectra requiring more averaging to obtain representative spectra of tissue types, and (3) the phosphate bands were generally higher in absorbance in the transflection measurements compared to the transmission ones. The amide I shift and the larger spectral variance was consistent with results obtained in previous studies where the EFWS was present. The findings indicate that artifacts resulting from transflection measurements were small but consistent across the tissue, and therefore the use of transflection measurements could be employed for disease diagnosis. Accordingly, we recommend a straightforward multivariate comparison of images from transmission and transflection measurements in a combined data matrix obtained from adjacent sections of the tissue as a useful preliminary study for establishing the impact of the EFWS on the samples, before considering the routine use of transflection substrates for any new tissue studied.
Pub.: 20 Feb '15, Pinned: 25 Aug '17
Abstract: New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.
Pub.: 22 Dec '15, Pinned: 25 Aug '17
Abstract: Fourier Transform Infrared (FTIR) and Raman imaging offer complementary information about the spatial location of molecules within cells. In this paper we investigate the integration of both imaging modalities in an extended image containing unique FTIR and Raman spectra for each pixel. Two types of cells were investigated: red blood cells infected with the Plasmodium falciparum parasite and Micrasterias, a desmid microalgal species. The microscope configuration and pixel size were selected specifically for acquiring images with the same pixel size, and samples were fixed to a sample holder in order to measure the same cells with the same orientation. Images were first analysed individually and then registered in an extended matrix containing an FTIR and a Raman spectra for each pixel. The results indicated that the combination of both techniques provide complementary information not evident in the analysis of individual images. The assignment of haemozoin FTIR bands from malaria trophozoites was only possible after correlating the FTIR spectrum with the Raman spectrum. The correlation of the Raman and FTIR spectral variables using statistical heterospectroscopy (SHY) enabled the assignment of overlapping lipid and carbohydrate bands. In summary, although the approach can be computing intensive the complementarity of the two techniques in terms of pixel resolution, signal to noise ratio and the assignment of vibrational modes makes this a powerful approach to diagnostic imaging.
Pub.: 01 Aug '16, Pinned: 25 Aug '17
Abstract: Fourier transform infrared spectroscopy (FTIR) imaging has been applied to investigate biochemical differences between salivary glands from control and hypertensive rats. Male Sprague-Dawley rats were divided into two groups including a control group and another hypertension group that were treated orally, with N-nitro-l-arginine methyl ester (l-NAME) via drinking water for 3 weeks to develop hypertension. In the control group, rats were treated with only drinking water for 3 weeks. The formalin-fixed paraffin embedded tissue specimens from submandibular and sublingual glands were analysed with a FTIR focal plane array imaging spectrometer and multi-composite images of all tissue sections were analysed simultaneously using Unsupervised Hierarchical Cluster Analysis (UHCA) and the extracted spectra were further analysed using Partial Least Squares Discriminant Analysis (PLS-DA). In general, hypertension affected salivary gland tissues were characterised by higher concentrations of triglycerides as evidenced by an increase in the 1745 cm(-1) band. Higher concentrations of carbohydrates and proteins were also observed in the hypertensive group along with a decrease in bands associated with nucleic acids. PLS-DA scores plots provided good differentiation in sublingual gland tissues between control (n = 3734 spectra) and hypertension (n = 4538) and also in submandibular gland tissues between control (n = 5051) and hypertension (n = 4408). We have shown that FTIR imaging can be used to differentiate the macromolecular information between physiological and pathological conditions in tissue biopsy specimens. In the next phase, we will investigate the infrared predictive markers of hypertension in biofluids including serum and saliva using attenuated total refection spectroscopy.
Pub.: 06 Feb '17, Pinned: 25 Aug '17
Abstract: Dengue fever is the most common mosquito transmitted viral infection afflicting humans, estimated to generate around 390 million infections each year in over 100 countries. The introduction of the endosymbiotic bacterium Wolbachia into Aedes aegypti mosquitoes has the potential to greatly reduce the public health burden of the disease. This approach requires extensive PCR (Polymerase Chain Reaction) testing of the Wolbachia-infection status of mosquitoes in areas where Wolbachia-A. aegypti are released. Here we report the first example of small organism mid-infrared spectroscopy where we have applied Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy and multivariate modelling methods to determine sex, age and the presence of Wolbachia (wMel strain) in laboratory mosquitoes and sex and age in field mosquitoes. The prediction errors using Partial Least Squares Discriminant Analysis (PLS-DA) discrimination models for laboratory studies on independent test sets ranged from 0 to 3% for age & sex grading, and 3 to 5% for Wolbachia infection diagnosis using dry mosquito abdomens while field study results using an Artificial neural network yielded a 10 % error. The application of FTIR analysis is inexpensive, easy to use, portable, and shows significant potential to replace the reliance on more expensive and laborious PCR assays.
Pub.: 24 Mar '17, Pinned: 25 Aug '17
Abstract: Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways.
Pub.: 03 Jun '17, Pinned: 25 Aug '17
Abstract: Attenuated total reflectance (ATR) infrared spectroscopy of dried organic extracts of serum samples has been evaluated as a fast method for the determination of triglycerides, cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL). After careful selection of solvents based on green parameters, serum samples were extracted using hexane-isopropanol and ethyl acetate-ethanol mixtures. Microscopy studies and comparison with standard spectra were performed in order to investigate whether the proposed methodology is suitable for the quantification of lipids in serum samples. The results of these preliminary studies confirmed that the variations in the IR spectra of sample extracts could be related quantitatively to variations in the concentrations of the target analytes. Then, ATR spectra of the dried sample extracts were obtained and direct measurement of the spectra were carried out and modelled using partial least squares (PLS) and reference concentrations. PLS models obtained from the extracts of the two mixtures were compared with those obtained from direct measurement of sera samples. The prediction errors obtained using the proposed approach were considerably (between 27 and 72%) better than those obtained by the direct measurements of sera. For triglycerides and cholesterol relative errors below 9% and 12% respectively were obtained with this method, which are comparable to the tolerance for the errors of the control analysis established at the hospital. For HDL and LDL, the errors found were between 18 and 20%. The incorporation of a preprocessing extraction step, involves time and solvent consumption. However, the results obtained provide evidence that the proposed method provides, in a few minutes and using simple instrumentation and with minimum cost, important information about the lipidic profile of patients sera at a good screening confidence level.
Pub.: 21 Nov '13, Pinned: 25 Aug '17
Abstract: Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) has the potential to become a new diagnostic tool for malaria and other diseases. For point-of-care testing, the use of ATR-FTIR in malaria diagnosis enables the analysis of blood in the aqueous state, which represents an enormous advantage by minimising the sample preparation by removing the need for cell fixation. Here we report the quantification of malaria parasitemia in human RBCs in their normal physiological aqueous state. A potential confounding variable for spectroscopic measurements performed on blood are the various anticoagulants that are required to prevent clotting. Accordingly, we tested the effects of 3 common anticoagulants; Sodium Citrate (SC), Potassium Ethylenediaminetetraacetic Acid (EDTA) and lithium heparin on plasma and whole blood in the aqueous and dry phase. Principal Component Analysis (PCA) revealed the model was heavily influenced by the anticoagulants in the case of dry samples, however, in aqueous whole blood samples, the effect was less pronounced as the water in the sample presumably diluted the amount of anticoagulant in contact with the ATR crystal. The possible influence of the anticoagulant effect on the ability to quantify parasitemia levels was tested using Partial Least Squares Regression Analysis (PLS-R). There was no influence of anticoagulants on quantification in the 0-1% range, however attempts to quantify at lower levels (0-0.1%) was best achieved with heparin compared to the other two anticoagulants. The results demonstrate ability to diagnose malaria using ATR-FTIR spectroscopy using wet RBC samples as well as underscoring the desirability to perform wet measurements as these minimise the possible confounding influence of anticoagulants used in blood collection.
Pub.: 07 Dec '16, Pinned: 25 Aug '17
Abstract: New diagnostic tools that can detect malaria parasites in conjunction with other diagnostic parameters are urgently required. In this study, Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy in combination with Partial Least Square Discriminant Analysis (PLS-DA) and Partial Least Square Regression (PLS-R) have been applied as a point-of-care test for identifying malaria parasites, blood glucose and urea levels in whole blood samples from thick blood films on glass slides. The specificity for the PLS-DA was found to be 98 % for parasitemia levels >0.5% but a rather low sensitivity of 70 % was achieved because of the small number of negative samples in the model. In PLS-R the Root Mean Square Error of Cross Validation (RMSECV) for parasite concentration (0-5%) was 0.58%. Similarly, for glucose (0-400 mg/dL) and urea (0-250mg/dL) spiked samples, relative RMSECVs were 16% and 17%, respectively. The method reported here is the first example of multi-analyte/disease diagnosis using ATR-FTIR spectroscopy, which in this case, enabled the simultaneous quantification of glucose and urea analytes along with malaria parasitemia quantification using one spectrum obtained from a single drop of blood on a glass microscope slide.
Pub.: 15 Apr '17, Pinned: 29 Jun '17