The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. Thes... more The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. These tests often need to be conducted frequently and quickly, but the logistics of sample collection and processing not only delays the test result, but also puts a strain on the healthcare system due to the sheer volume of tests that need to be performed. The advent of microfluidics has made the processing of samples quick and reliable, with little or no skill required on the user’s part. However, while several microfluidic devices have been demonstrated for plasma separation, none of them have validated the chemical integrity of the sample post-process. Here, we present Haemoprocessor: a portable, robust, open-fluidic system that utilizes Travelling Surface Acoustic Waves (TSAW) with the expression of overtones to separate plasma from 20× diluted human blood within a span of 2 min to achieve 98% RBC removal. The plasma and red blood cell separation quality/integrity was validated through ...
Serum is an important candidate in proteomics analysis as it potentially carries key markers on h... more Serum is an important candidate in proteomics analysis as it potentially carries key markers on health status and disease progression. However, several important diagnostic markers found in the circulatory proteome and the low-molecular-weight (LMW) peptidome have become analytically challenging due to the high dynamic concentration range of the constituent protein/peptide species in serum. Herein, we propose a novel approach to improve the limit of detection (LoD) of LMW amino acids by combining mid-IR (MIR) and near-IR spectroscopic data using glycine as a model LMW analyte. This is the first example of near-IR spectroscopy applied to elucidate the detection limit of LMW components in serum; moreover, it is the first study of its kind to combine mid-infrared (25–2.5 μm) and near-infrared (2500–800 nm) to detect an analyte in serum. First, we evaluated the prediction model performance individually with MIR (ATR-FTIR) and NIR spectroscopic methods using partial least squares regress...
Research groups around the world are studying the spatial location and distribution of molecules ... more Research groups around the world are studying the spatial location and distribution of molecules within cells using an increasing number of analytical techniques such as infrared (IR), Raman and X-ray fluorescence (XRF) spectroscopy. The information obtained from these techniques in terms of lipids, proteins and the general metabolome is complementary, but commonly the analysis of the data is performed individually on each technique. These three techniques are based on different interactions of the sample with light with different energy and wavelengths, leading to dissimilarities in the spectral features offered by each technique. Table 1 summarises the main features of Raman and IR microspectroscopy, both representing vibrational spectroscopic methods. Raman spectroscopy is a scattering technique, in which energy is transmitted from a photon to a molecule, resulting in a shift in the wavelength of the incident light beam. Fourier transform (FT)-IR spectroscopy, on the other hand, ...
Coupling synchrotron IR beam to an ATR element enhances spatial resolution suited for high-resolu... more Coupling synchrotron IR beam to an ATR element enhances spatial resolution suited for high-resolution single cell analysis in biology, medicine and environmental science.
A spectroscopic technique is presented that is able to identify rapid changes in the bending modu... more A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids,...
Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complicati... more Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology t...
New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of... more 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,...
Journal of the Royal Society, Interface / the Royal Society, Jan 6, 2015
Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cyt... more Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2...
The strong enhancement when applying near-infrared excitation wavelengths, of totally symmetric m... more The strong enhancement when applying near-infrared excitation wavelengths, of totally symmetric modes of heme derivatives including β-hematin (malaria pigment), was hypothesized to be due to supramolecular interaction. Specifically, the intensity of the electron density or oxidation state marker band, ν4, is in part strongly affected by CH(((X hydrogen bonding interactions when X is an electron-donating entity. Chlorotetra (p-methoxyphenyl) porphyrinatoiron (III), Fe (TMPP)Cl, is a model compound that supports this hypothesis. The single crystal X-ray structure of Fe (TMPP)Cl, recrystallized by vapor diffusion of diethyl ether into dichloromethane solution, was determined. There are πpor···πpor interactions and numerous additional supramolecular interactions in the form of CH···Cl and CH···O, and CH···π hydrogen bond interactions. The corresponding resonance Raman intensity of Fe (TMPP)Cl exhibited great enhancement of the ν4 band when using 780 and 830 nm laser sources supporting t...
Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood ... more Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood with the aim of developing an online clinical diagnostic and monitoring modality. Leucocyte adsorption onto the CaF(2) windows was observed over a period of several hours under continuous blood flow using a transmission cell of 30 mum path length. This build-up of cellular material on the windows is responsible for diminishing the sample path length under the flow conditions chosen. The adsorption dynamics have been characterised and their impact on glucose monitoring is reported. For short-term monitoring (<2 hours) a standard error of prediction of 11 mg/dL with human citrated blood samples from three different subjects was achieved. Furthermore, the leucocyte build-up was also reported for porcine EDTA blood monitoring. Consequences and testing opportunities with regard to the first stages in the immune cell reaction to the exposure of body-foreign materials to anticoagulated whole blood are discussed.
Permutation testing in the evaluation of the statistical significance for infrared based classifi... more Permutation testing in the evaluation of the statistical significance for infrared based classification of biological samples.
The human hair follicle mesenchyme contains multipotent stem cells[1-3] that have important roles... more The human hair follicle mesenchyme contains multipotent stem cells[1-3] that have important roles in dermal reconstruction during cutaneous wound healing[1,4]. The purification and characterisation of the stem cells are met with difficulties, particularly due to the lack of specific antigens for ...
Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were use... more Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were used to compare chloroquine (CQ)-treated and untreated cultured Plasmodium falciparum-infected human red blood cells (iRBCs).
Recently a resonant Mie scattering (RMieS) correction approach has been developed and demonstrate... more Recently a resonant Mie scattering (RMieS) correction approach has been developed and demonstrated to be effective for removing the baseline distortions that compromise the raw data in individual spectra. In this paper RMieS correction is extended to FTIR images of a tissue section from biopsy of the human cervical transformation zone and a coronal tissue section of a Wistar rat brain and compared to the uncorrected images. It is shown that applying RMieS correction to FTIR images a) removes baseline distortions from the image spectra and thus reveals previously hidden information on spatial variation of chemical contents within the tissue and b) can lead to improved automatic tissue feature classification through multivariate cluster analysis.
ABSTRACT Prostate cancer cells derived from brain metastasis (DU-145 cell line) were used as a mo... more ABSTRACT Prostate cancer cells derived from brain metastasis (DU-145 cell line) were used as a model. Single cells were irradiated with a controlled number (20–4000) of 1 MeV protons from the Cracow microbe. After fixation, irradiated and control cells were investigated using FTIR microspectroscopy. Quantitative and qualitative changes in phosphodiester region associated with DNA radiation damage, were observed. a b s t r a c t The effect of ionizing radiation on single cells using a proton source was investigated using Fourier transform infrared (FTIR) microspectroscopy. The prostate cancer cells (DU-145) were irradiated by a specific number (50, 200, 400, 2000 and 4000) of protons per cell. Next after fixing the cells with 70% ethanol micro-FTIR spectra were obtained using both: (a) the synchrotron radiation source with a Mercury–Cadmium–Telluride (MCT) detector and (b) a globar source with a focal plane array (FPA) detector. FTIR spectra obtained from both instrumental configurations were analyzed independently to investigate the changes in the DNA phosphodiester region (1150–950 cm −1) of irradiated and control (untreated by ionizing radiation) cells. A Principal Component Analysis (PCA) scores plot revealed distinct clusters for all groups of irradiated cells, even for those irradiated by the smallest dose of protons. The dose-dependent changes in the relative intensities of DNA peak at 970 cm −1 (ribose-phosphate skeletal motions), along with a shift of the O–P–O band corresponding to the symmetric phosphodiester stretching mode at 1090 cm −1 were observed. The results demonstrate that FTIR spectroscopy is a promising tool to investigate DNA damage in single cells and may become an important tool in assessing cell damage following radiotherapy.
The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. Thes... more The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. These tests often need to be conducted frequently and quickly, but the logistics of sample collection and processing not only delays the test result, but also puts a strain on the healthcare system due to the sheer volume of tests that need to be performed. The advent of microfluidics has made the processing of samples quick and reliable, with little or no skill required on the user’s part. However, while several microfluidic devices have been demonstrated for plasma separation, none of them have validated the chemical integrity of the sample post-process. Here, we present Haemoprocessor: a portable, robust, open-fluidic system that utilizes Travelling Surface Acoustic Waves (TSAW) with the expression of overtones to separate plasma from 20× diluted human blood within a span of 2 min to achieve 98% RBC removal. The plasma and red blood cell separation quality/integrity was validated through ...
Serum is an important candidate in proteomics analysis as it potentially carries key markers on h... more Serum is an important candidate in proteomics analysis as it potentially carries key markers on health status and disease progression. However, several important diagnostic markers found in the circulatory proteome and the low-molecular-weight (LMW) peptidome have become analytically challenging due to the high dynamic concentration range of the constituent protein/peptide species in serum. Herein, we propose a novel approach to improve the limit of detection (LoD) of LMW amino acids by combining mid-IR (MIR) and near-IR spectroscopic data using glycine as a model LMW analyte. This is the first example of near-IR spectroscopy applied to elucidate the detection limit of LMW components in serum; moreover, it is the first study of its kind to combine mid-infrared (25–2.5 μm) and near-infrared (2500–800 nm) to detect an analyte in serum. First, we evaluated the prediction model performance individually with MIR (ATR-FTIR) and NIR spectroscopic methods using partial least squares regress...
Research groups around the world are studying the spatial location and distribution of molecules ... more Research groups around the world are studying the spatial location and distribution of molecules within cells using an increasing number of analytical techniques such as infrared (IR), Raman and X-ray fluorescence (XRF) spectroscopy. The information obtained from these techniques in terms of lipids, proteins and the general metabolome is complementary, but commonly the analysis of the data is performed individually on each technique. These three techniques are based on different interactions of the sample with light with different energy and wavelengths, leading to dissimilarities in the spectral features offered by each technique. Table 1 summarises the main features of Raman and IR microspectroscopy, both representing vibrational spectroscopic methods. Raman spectroscopy is a scattering technique, in which energy is transmitted from a photon to a molecule, resulting in a shift in the wavelength of the incident light beam. Fourier transform (FT)-IR spectroscopy, on the other hand, ...
Coupling synchrotron IR beam to an ATR element enhances spatial resolution suited for high-resolu... more Coupling synchrotron IR beam to an ATR element enhances spatial resolution suited for high-resolution single cell analysis in biology, medicine and environmental science.
A spectroscopic technique is presented that is able to identify rapid changes in the bending modu... more A spectroscopic technique is presented that is able to identify rapid changes in the bending modulus and fluidity of vesicle lipid bilayers on the micrometer scale, and distinguish between the presence and absence of heterogeneities in lipid-packing order. Individual unilamellar vesicles have been isolated using laser tweezers and, by measuring the intensity modulation of elastic back-scattered light, changes in the biophysical properties of lipid bilayers were revealed. Our approach offers unprecedented temporal resolution and, uniquely, physical transformations of lipid bilayers can be monitored on a length scale of micrometers. As an example, the deformation of a membrane bilayer following the gel-to-fluid phase transition in a pure phospholipid vesicle was observed to take place across an interval of 54 ± 5 ms corresponding to an estimated full-width of only ~1 m°C. Dynamic heterogeneities in packing order were detected in mixed-lipid bilayers. Using a ternary mixture of lipids,...
Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complicati... more Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology t...
New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of... more 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,...
Journal of the Royal Society, Interface / the Royal Society, Jan 6, 2015
Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cyt... more Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2...
The strong enhancement when applying near-infrared excitation wavelengths, of totally symmetric m... more The strong enhancement when applying near-infrared excitation wavelengths, of totally symmetric modes of heme derivatives including β-hematin (malaria pigment), was hypothesized to be due to supramolecular interaction. Specifically, the intensity of the electron density or oxidation state marker band, ν4, is in part strongly affected by CH(((X hydrogen bonding interactions when X is an electron-donating entity. Chlorotetra (p-methoxyphenyl) porphyrinatoiron (III), Fe (TMPP)Cl, is a model compound that supports this hypothesis. The single crystal X-ray structure of Fe (TMPP)Cl, recrystallized by vapor diffusion of diethyl ether into dichloromethane solution, was determined. There are πpor···πpor interactions and numerous additional supramolecular interactions in the form of CH···Cl and CH···O, and CH···π hydrogen bond interactions. The corresponding resonance Raman intensity of Fe (TMPP)Cl exhibited great enhancement of the ν4 band when using 780 and 830 nm laser sources supporting t...
Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood ... more Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood with the aim of developing an online clinical diagnostic and monitoring modality. Leucocyte adsorption onto the CaF(2) windows was observed over a period of several hours under continuous blood flow using a transmission cell of 30 mum path length. This build-up of cellular material on the windows is responsible for diminishing the sample path length under the flow conditions chosen. The adsorption dynamics have been characterised and their impact on glucose monitoring is reported. For short-term monitoring (<2 hours) a standard error of prediction of 11 mg/dL with human citrated blood samples from three different subjects was achieved. Furthermore, the leucocyte build-up was also reported for porcine EDTA blood monitoring. Consequences and testing opportunities with regard to the first stages in the immune cell reaction to the exposure of body-foreign materials to anticoagulated whole blood are discussed.
Permutation testing in the evaluation of the statistical significance for infrared based classifi... more Permutation testing in the evaluation of the statistical significance for infrared based classification of biological samples.
The human hair follicle mesenchyme contains multipotent stem cells[1-3] that have important roles... more The human hair follicle mesenchyme contains multipotent stem cells[1-3] that have important roles in dermal reconstruction during cutaneous wound healing[1,4]. The purification and characterisation of the stem cells are met with difficulties, particularly due to the lack of specific antigens for ...
Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were use... more Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were used to compare chloroquine (CQ)-treated and untreated cultured Plasmodium falciparum-infected human red blood cells (iRBCs).
Recently a resonant Mie scattering (RMieS) correction approach has been developed and demonstrate... more Recently a resonant Mie scattering (RMieS) correction approach has been developed and demonstrated to be effective for removing the baseline distortions that compromise the raw data in individual spectra. In this paper RMieS correction is extended to FTIR images of a tissue section from biopsy of the human cervical transformation zone and a coronal tissue section of a Wistar rat brain and compared to the uncorrected images. It is shown that applying RMieS correction to FTIR images a) removes baseline distortions from the image spectra and thus reveals previously hidden information on spatial variation of chemical contents within the tissue and b) can lead to improved automatic tissue feature classification through multivariate cluster analysis.
ABSTRACT Prostate cancer cells derived from brain metastasis (DU-145 cell line) were used as a mo... more ABSTRACT Prostate cancer cells derived from brain metastasis (DU-145 cell line) were used as a model. Single cells were irradiated with a controlled number (20–4000) of 1 MeV protons from the Cracow microbe. After fixation, irradiated and control cells were investigated using FTIR microspectroscopy. Quantitative and qualitative changes in phosphodiester region associated with DNA radiation damage, were observed. a b s t r a c t The effect of ionizing radiation on single cells using a proton source was investigated using Fourier transform infrared (FTIR) microspectroscopy. The prostate cancer cells (DU-145) were irradiated by a specific number (50, 200, 400, 2000 and 4000) of protons per cell. Next after fixing the cells with 70% ethanol micro-FTIR spectra were obtained using both: (a) the synchrotron radiation source with a Mercury–Cadmium–Telluride (MCT) detector and (b) a globar source with a focal plane array (FPA) detector. FTIR spectra obtained from both instrumental configurations were analyzed independently to investigate the changes in the DNA phosphodiester region (1150–950 cm −1) of irradiated and control (untreated by ionizing radiation) cells. A Principal Component Analysis (PCA) scores plot revealed distinct clusters for all groups of irradiated cells, even for those irradiated by the smallest dose of protons. The dose-dependent changes in the relative intensities of DNA peak at 970 cm −1 (ribose-phosphate skeletal motions), along with a shift of the O–P–O band corresponding to the symmetric phosphodiester stretching mode at 1090 cm −1 were observed. The results demonstrate that FTIR spectroscopy is a promising tool to investigate DNA damage in single cells and may become an important tool in assessing cell damage following radiotherapy.
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