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1.
Article in English | MEDLINE | ID: mdl-38479560

ABSTRACT

PURPOSE: Neutron capture enhanced particle therapy (NCEPT) is a proposed augmentation of charged particle therapy that exploits thermal neutrons generated internally, within the treatment volume via nuclear fragmentation, to deliver a biochemically targeted radiation dose to cancer cells. This work is the first experimental demonstration of NCEPT, performed using both carbon and helium ion beams with 2 different targeted neutron capture agents (NCAs). METHODS AND MATERIALS: Human glioblastoma cells (T98G) were irradiated by carbon and helium ion beams in the presence of NCAs [10B]-BPA and [157Gd]-DOTA-TPP. Cells were positioned within a polymethyl methacrylate phantom either laterally adjacent to or within a 100 × 100 × 60 mm spread out Bragg peak (SOBP). The effect of NCAs and location relative to the SOBP on the cells was measured by cell growth and survival assays in 6 independent experiments. Neutron fluence within the phantom was characterized by quantifying the neutron activation of gold foil. RESULTS: Cells placed inside the treatment volume reached 10% survival by 2 Gy of carbon or 2 to 3 Gy of helium in the presence of NCAs compared with 5 Gy of carbon and 7 Gy of helium with no NCA. Cells placed adjacent to the treatment volume showed a dose-dependent decrease in cell growth when treated with NCAs, reaching 10% survival by 6 Gy of carbon or helium (to the treatment volume), compared with no detectable effect on cells without NCA. The mean thermal neutron fluence at the center of the SOBP was approximately 2.2 × 109 n/cm2/Gy (relative biological effectiveness) for the carbon beam and 5.8 × 109 n/cm2/Gy (relative biological effectiveness) for the helium beam and gradually decreased in all directions. CONCLUSIONS: The addition of NCAs to cancer cells during carbon and helium beam irradiation has a measurable effect on cell survival and growth in vitro. Through the capture of internally generated neutrons, NCEPT introduces the concept of a biochemically targeted radiation dose to charged particle therapy. NCEPT enables the established pharmaceuticals and concepts of neutron capture therapy to be applied to a wider range of deeply situated and diffuse tumors, by targeting this dose to microinfiltrates and cells outside of defined treatment regions. These results also demonstrate the potential for NCEPT to provide an increased dose to tumor tissue within the treatment volume, with a reduction in radiation doses to off-target tissue.

2.
Biosensors (Basel) ; 12(12)2022 Nov 24.
Article in English | MEDLINE | ID: mdl-36551040

ABSTRACT

Phase imaging of biochemical samples has been demonstrated for the first time at the Infrared Microspectroscopy (IRM) beamline of the Australian Synchrotron using the usually discarded near-IR (NIR) region of the synchrotron-IR beam. The synchrotron-IR beam at the Australian Synchrotron IRM beamline has a unique fork shaped intensity distribution as a result of the gold coated extraction mirror shape, which includes a central slit for rejection of the intense X-ray beam. The resulting beam configuration makes any imaging task challenging. For intensity imaging, the fork shaped beam is usually tightly focused to a point on the sample plane followed by a pixel-by-pixel scanning approach to record the image. In this study, a pinhole was aligned with one of the lobes of the fork shaped beam and the Airy diffraction pattern was used to illuminate biochemical samples. The diffracted light from the samples was captured using a NIR sensitive lensless camera. A rapid phase-retrieval algorithm was applied to the recorded intensity distributions to reconstruct the phase information. The preliminary results are promising to develop multimodal imaging capabilities at the IRM beamline of the Australian Synchrotron.


Subject(s)
Multimodal Imaging , Synchrotrons , Australia , Algorithms
3.
J Synchrotron Radiat ; 28(Pt 5): 1616-1619, 2021 Sep 01.
Article in English | MEDLINE | ID: mdl-34475308

ABSTRACT

The Infrared Microspectroscopy Beamline at the Australian Synchrotron is equipped with a Fourier transform infrared (FTIR) spectrometer, which is coupled with an infrared (IR) microscope and a choice of two detectors: a single-point narrow-band mercury cadmium telluride (MCT) detector and a 64 × 64 multi-pixel focal plane array (FPA) imaging detector. A scanning-based point-by-point mapping method is commonly used with a tightly focused synchrotron IR beam at the sample plane, using an MCT detector and a matching 36× IR reflecting objective and condenser (NA = 0.5), which is time consuming. In this study, the beam size at the sample plane was increased using a 15× objective and the spatio-spectral aberrations were investigated. A correlation-based semi-synthetic computational optical approach was applied to assess the possibilities of exploiting the aberrations to perform rapid imaging rather than a mapping approach.

4.
Angew Chem Int Ed Engl ; 60(31): 17102-17107, 2021 07 26.
Article in English | MEDLINE | ID: mdl-34043272

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Saliva/chemistry , Animals , Chlorocebus aethiops , Cohort Studies , Discriminant Analysis , Humans , Least-Squares Analysis , Monte Carlo Method , Point-of-Care Testing , Proof of Concept Study , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling , Spectrophotometry, Infrared , Vero Cells
5.
Analyst ; 146(11): 3516-3525, 2021 Jun 07.
Article in English | MEDLINE | ID: mdl-33881057

ABSTRACT

Visualising direct biochemical markers of cell physiology and disease pathology at the sub-cellular level is an ongoing challenge in the biological sciences. A suite of microscopies exists to either visualise sub-cellular architecture or to indirectly view biochemical markers (e.g. histochemistry), but further technique developments and innovations are required to increase the range of biochemical parameters that can be imaged directly, in situ, within cells and tissue. Here, we report our continued advancements in the application of synchrotron radiation attenuated total reflectance Fourier transform infrared (SR-ATR-FTIR) microspectroscopy to study sub-cellular biochemistry. Our recent applications demonstrate the much needed capability to map or image directly sub-cellular protein aggregates within degenerating neurons as well as lipid inclusions within bacterial cells. We also characterise the effect of spectral acquisition parameters on speed of data collection and the associated trade-offs between a realistic experimental time frame and spectral/image quality. Specifically, the study highlights that the choice of 8 cm-1 spectral resolutions provide a suitable trade-off between spectral quality and collection time, enabling identification of important spectroscopic markers, while increasing image acquisition by ∼30% (relative to 4 cm-1 spectral resolution). Further, this study explores coupling a focal plane array detector with SR-ATR-FTIR, revealing a modest time improvement in image acquisition time (factor of 2.8). Such information continues to lay the foundation for these spectroscopic methods to be readily available for, and adopted by, the biological science community to facilitate new interdisciplinary endeavours to unravel complex biochemical questions and expand emerging areas of study.


Subject(s)
Protein Aggregates , Synchrotrons , Lipids , Proteins , Spectroscopy, Fourier Transform Infrared
6.
Sci Rep ; 10(1): 11713, 2020 07 16.
Article in English | MEDLINE | ID: mdl-32678217

ABSTRACT

The ovalbumin-induced (OVA) chronic allergic airways murine model is a well-established model for investigating pre-clinical therapies for chronic allergic airways diseases, such as asthma. Here, we examined the effects of several experimental compounds with potential anti-asthmatic effects including resveratrol (RV), relaxin (RLN), L-sulforaphane (LSF), valproic acid (VPA), and trichostatin A (TSA) using both a prevention and reversal model of chronic allergic airways disease. We undertook a novel analytical approach using focal plane array (FPA) and synchrotron Fourier-transform infrared (S-FTIR) microspectroscopic techniques to provide new insights into the mechanisms of action of these experimental compounds. Apart from the typical biological effects, S-FTIR microspectroscopy was able to detect changes in nucleic acids and protein acetylation. Further, we validated the reduction in collagen deposition induced by each experimental compound evaluated. Although this has previously been observed with conventional histological methods, the S-FTIR technique has the advantage of allowing identification of the type of collagen present. More generally, our findings highlight the potential utility of S-FTIR and FPA-FTIR imaging techniques in enabling a better mechanistic understanding of novel asthma therapeutics.


Subject(s)
Anti-Asthmatic Agents/administration & dosage , Asthma/drug therapy , Hydroxamic Acids/administration & dosage , Isothiocyanates/administration & dosage , Relaxin/administration & dosage , Resveratrol/administration & dosage , Valproic Acid/administration & dosage , Animals , Asthma/chemically induced , Chronic Disease/drug therapy , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Female , Mice , Mice, Inbred BALB C , Ovalbumin/adverse effects , Spectroscopy, Fourier Transform Infrared/methods , Sulfoxides , Synchrotrons , Treatment Outcome
7.
J Mol Graph Model ; 97: 107568, 2020 06.
Article in English | MEDLINE | ID: mdl-32097886

ABSTRACT

Streptococcus pneumoniae infection can lead to pneumococcal disease, a major cause of mortality in children under the age of five years. In low- and middle-income country settings where pneumococcal disease burden is high, vaccine use is low and widespread antibiotic use has led to increased rates of multi-drug resistant pneumococci. l-sulforaphane (LSF), derived from broccoli and other cruciferous vegetables, has established anti-inflammatory, antioxidant, and anti-microbial properties. Hence, we sought to investigate the potential role of LSF against pneumococcal infection. Using a combination of in vitro and computational methods, the results showed that LSF and relevant metabolites had a potential to reduce pneumococcal adherence through modulation of host receptors, regulation of inflammation, or through direct modification of bacterial factors. Treatment with LSF and metabolites reduced pneumococcal adherence to respiratory epithelial cells. Synchrotron-Fourier transform infrared microspectroscopy (S-FTIR) revealed biochemical changes in protein and lipid profiles of lung epithelial cells following treatment with LSF or metabolites. Molecular docking studies of 116 pneumococcal and 89 host factors revealed a potent effect for the metabolite LSF-glutathione (GSH). A comprehensive list of factors involved in interactions between S. pneumoniae and host cells was compiled to construct a bacterium and host interaction network. Network analysis revealed plasminogen, fibronectin, and RrgA as key factors involved in pneumococcal-host interactions. Therefore, we propose that these constitute critical targets for direct inhibition by LSF and/or metabolites, which may disrupt pneumococcal-host adherence. Overall, our findings further enhance understanding of the potential role of LSF to modulate pneumococcal-host dynamics.


Subject(s)
Streptococcus pneumoniae , Synchrotrons , Child , Child, Preschool , Humans , Isothiocyanates , Molecular Docking Simulation , Spectroscopy, Fourier Transform Infrared , Sulfoxides
9.
Nat Commun ; 10(1): 3436, 2019 07 31.
Article in English | MEDLINE | ID: mdl-31366886

ABSTRACT

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.


Subject(s)
Autophagy/physiology , Bone Diseases, Developmental/genetics , Calcification, Physiologic/physiology , Ephrin-B2/metabolism , rho GTP-Binding Proteins/metabolism , Animals , Autophagosomes/physiology , Autophagy/genetics , Bone Diseases, Developmental/pathology , Bone Remodeling/physiology , Cell Line , Ephrin-B2/genetics , Mice , Mice, Inbred C57BL , Osteocytes/metabolism , Osteocytes/physiology , RNA Interference , RNA, Small Interfering/genetics , rhoA GTP-Binding Protein
10.
Calcif Tissue Int ; 103(6): 625-637, 2018 12.
Article in English | MEDLINE | ID: mdl-30019315

ABSTRACT

Bone is formed by deposition of a collagen-containing matrix (osteoid) that hardens over time as mineral crystals accrue and are modified; this continues until bone remodeling renews that site. Pharmacological agents for osteoporosis differ in their effects on bone remodeling, and we hypothesized that they may differently modify bone mineral accrual. We, therefore, assessed newly formed bone in mature ovariectomized rabbits treated with the anti-resorptive bisphosphonate alendronate (ALN-100µ g/kg/2×/week), the anabolic parathyroid hormone (PTH (1-34)-15µ g/kg/5×/week), or the experimental anti-resorptive odanacatib (ODN 7.5 µM/day), which suppresses bone resorption without suppressing bone formation. Treatments were administered for 10 months commencing 6 months after ovariectomy (OVX). Strength testing, histomorphometry, and synchrotron Fourier-transform infrared microspectroscopy were used to measure bone strength, bone formation, and mineral accrual, respectively, in newly formed endocortical and intracortical bone. In Sham and OVX endocortical and intracortical bone, three modifications occurred as the bone matrix aged: mineral accrual (increase in mineral:matrix ratio), carbonate substitution (increase in carbonate:mineral ratio), and collagen molecular compaction (decrease in amide I:II ratio). ALN suppressed bone formation but mineral accrued normally at those sites where bone formation occurred. PTH stimulated bone formation on endocortical, periosteal, and intracortical bone surfaces, but mineral accrual and carbonate substitution were suppressed, particularly in intracortical bone. ODN treatment did not suppress bone formation, but newly deposited endocortical bone matured more slowly with ODN, and ODN-treated intracortical bone had less carbonate substitution than controls. In conclusion, these agents differ in their effects on the bone matrix. While ALN suppresses bone formation, it does not modify bone mineral accrual in endocortical or intracortical bone. While ODN does not suppress bone formation, it slows matrix maturation. PTH stimulates modelling-based bone formation not only on endocortical and trabecular surfaces, but may also do so in intracortical bone; at this site, new bone deposited contains less mineral than normal.


Subject(s)
Alendronate/pharmacology , Biphenyl Compounds/pharmacology , Bone Density Conservation Agents/pharmacology , Bone and Bones/drug effects , Parathyroid Hormone/pharmacology , Animals , Bone Remodeling/drug effects , Bone and Bones/physiology , Calcification, Physiologic/drug effects , Female , Osteogenesis/drug effects , Ovariectomy , Rabbits
11.
Sci Rep ; 7(1): 2649, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28572622

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.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/chemistry , Bezafibrate/chemistry , Bezafibrate/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Medroxyprogesterone/chemistry , Antineoplastic Combined Chemotherapy Protocols/pharmacology , HL-60 Cells , Humans , Medroxyprogesterone/pharmacology , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Synchrotrons
12.
Hell J Nucl Med ; 20 Suppl: 103-113, 2017.
Article in English | MEDLINE | ID: mdl-29324919

ABSTRACT

Asthma is a chronic respiratory disease characterised by airway inflammation, remodeling and hyperresponsiveness. The ability to replicate these asthma traits in the well-established ovalbumin induced chronic model of allergic airways disease is an important tool for asthma research and preclinical drug development. Here, spectra derived from focal plane array and Synchrotron-Fourier transform infrared maps were used to analyse biochemical changes in lung tissue from an ovalbumin-induced murine chronic allergic airways disease model. Analysis of the chemical maps resulted in distinct clusters and significant changes in the lipid and proteins regions of the spectra between the saline control and diseased lung tissue samples. Overall, the utilisation of conventional histological methodologies and Synchrotron infrared microspectroscopy has the ability to expand the characterisation of murine models of asthma.


Subject(s)
Asthma/immunology , Asthma/pathology , Ovalbumin/immunology , Spectroscopy, Fourier Transform Infrared/instrumentation , Synchrotrons , Animals , Asthma/diagnosis , Histology , Lung/immunology , Lung/pathology , Mice , Mice, Inbred BALB C
13.
Bone ; 93: 146-154, 2016 12.
Article in English | MEDLINE | ID: mdl-27686599

ABSTRACT

Intermittent administration of parathyroid hormone (PTH) is used to stimulate bone formation in patients with osteoporosis. A reduction in the degree of matrix mineralisation has been reported during treatment, which may reflect either production of undermineralised matrix or a greater proportion of new matrix within the bone samples assessed. To explore these alternatives, high resolution synchrotron-based Fourier Transform Infrared Microspectroscopy (sFTIRM) coupled with calcein labelling was used in a region of non-remodelling cortical bone to determine bone composition during anabolic PTH treatment compared with region-matched samples from controls. 8week old male C57BL/6 mice were treated with vehicle or 50µg/kg PTH, 5 times/week for 4weeks (n=7-9/group). Histomorphometry confirmed greater trabecular and periosteal bone formation and 3-point bending tests confirmed greater femoral strength in PTH-treated mice. Dual calcein labels were used to match bone regions by time-since-mineralisation (bone age) and composition was measured by sFTIRM in six 15µm2 regions at increasing depth perpendicular to the most immature bone on the medial periosteal edge; this allowed in situ measurement of progressive changes in bone matrix during its maturation. The sFTIRM method was validated in vehicle-treated bones where the expected progressive increases in mineral:matrix ratio and collagen crosslink type ratio were detected with increasing bone maturity. We also observed a gradual increase in carbonate content that strongly correlated with an increase in longitudinal stretch of the collagen triple helix (amide I:amide II ratio). PTH treatment did not alter the progressive changes in any of these parameters from the periosteal edge through to the more mature bone. These data provide new information about how the bone matrix matures in situ and confirm that bone deposited during PTH treatment undergoes normal collagen maturation and normal mineral accrual.


Subject(s)
Anabolic Agents/pharmacology , Bone Density/drug effects , Bone Development/drug effects , Bone Matrix/physiology , Parathyroid Hormone/pharmacology , Age Determination by Skeleton , Animals , Biomechanical Phenomena , Bone Matrix/drug effects , Calcification, Physiologic/drug effects , Cancellous Bone/anatomy & histology , Cancellous Bone/diagnostic imaging , Cancellous Bone/drug effects , Cancellous Bone/physiology , Collagen/metabolism , Male , Mice, Inbred C57BL , Microspectrophotometry , Minerals/metabolism , Organ Size/drug effects , Osteoblasts/cytology , Osteoblasts/drug effects , Periosteum/drug effects , Periosteum/physiology , Spectroscopy, Fourier Transform Infrared , Synchrotrons , Tibia/anatomy & histology , Tibia/diagnostic imaging , Tibia/physiology
14.
PLoS One ; 10(2): e0116491, 2015.
Article in English | MEDLINE | ID: mdl-25710811

ABSTRACT

Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.


Subject(s)
Artifacts , Histocytological Preparation Techniques/methods , Spectroscopy, Fourier Transform Infrared/methods , Animals , Myocardium/cytology , Rats
15.
Phys Chem Chem Phys ; 17(33): 21164-8, 2015 Sep 07.
Article in English | MEDLINE | ID: mdl-25491490

ABSTRACT

Surface enhanced Raman scattering (SERS) is a powerful tool with great potential to provide improved bio-sensing capabilities. The current 'gold-standard' method for diagnosis of malaria involves visual inspection of blood smears using light microscopy, which is time consuming and can prevent early diagnosis of the disease. We present a novel surface-enhanced Raman spectroscopy substrate based on gold-coated butterfly wings, which enabled detection of malarial hemozoin pigment within lysed blood samples containing 0.005% and 0.0005% infected red blood cells.


Subject(s)
Malaria/diagnosis , Nanostructures/chemistry , Plasmodium/isolation & purification , Spectrum Analysis, Raman , Wings, Animal/chemistry , Animals , Butterflies/physiology , Erythrocytes/parasitology , Gold/chemistry , Hemeproteins/analysis , Hemeproteins/chemistry , Humans , Malaria/parasitology , Nanostructures/ultrastructure , Plasmodium/metabolism
16.
Talanta ; 130: 39-48, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25159377

ABSTRACT

Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation.


Subject(s)
Biomarkers/analysis , Cell Differentiation , Chondrocytes/cytology , Mesenchymal Stem Cells/cytology , Spectroscopy, Fourier Transform Infrared/methods , Biomarkers/metabolism , Blotting, Western , Cells, Cultured , Discriminant Analysis , Humans , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
17.
Analyst ; 139(19): 4769-74, 2014 Oct 07.
Article in English | MEDLINE | ID: mdl-25055796

ABSTRACT

New methods are needed to rapidly identify malaria parasites in blood smears. The coupling of a Focal Plane Array (FPA) infrared microscope system to a synchrotron light source at IRENI enables rapid molecular imaging at high spatial resolution. The technique, in combination with hyper-spectral processing, enables imaging and diagnosis of early stage malaria parasites at the single cell level in a blood smear. The method relies on the detection of distinct lipid signatures associated with the different stages of the malaria parasite and utilises resonant Mie extended multiplicative scatter correction to pre-process the spectra followed by full bandwidth image deconvolution to resolve the single cells. This work demonstrates the potential of focal plane technology to diagnose single cells in a blood smear. Brighter laboratory based infrared sources, optical refinements and higher sensitive detectors will soon see the emergence of focal plane array imaging in the clinical environment.


Subject(s)
Malaria/diagnosis , Photomicrography , Spectroscopy, Fourier Transform Infrared , Erythrocytes/cytology , Erythrocytes/parasitology , Humans , Image Processing, Computer-Assisted , Malaria/parasitology , Plasmodium falciparum/immunology , Plasmodium falciparum/isolation & purification , Principal Component Analysis , Single-Cell Analysis , Tissue Array Analysis
18.
Analyst ; 139(17): 4200-9, 2014 Sep 07.
Article in English | MEDLINE | ID: mdl-24995477

ABSTRACT

SR-FTIR in combination with Principal Component Analysis (PCA) was applied to investigate macromolecular changes in a population of melanocytes and their extracted nuclei induced by environmentally relevant fluxes of UVR (Ultraviolet Radiation). Living cells and isolated cellular nuclei were investigated post-irradiation for three different irradiation dosages (130, 1505, 15,052 Jm(-2) UVR, weighted) after either 24 or 48 hours of incubation. DNA conformational changes were observed in cells exposed to an artificial UVR solar-simulator source as evidenced by a shift in the DNA asymmetric phosphodiester vibration from 1236 cm(-1) to 1242 cm(-1) in the case of the exposed cells and from 1225 cm(-1) to 1242 cm(-1) for irradiated nuclei. PCA Scores plots revealed distinct clustering of spectra from irradiated cells and nuclei from non-irradiated controls in response to the range of applied UVR radiation doses. 3D Raman confocal imaging in combination with k-means cluster analysis was applied to study the effect of the UVR radiation exposure on cellular nuclei. Chemical changes associated with apoptosis were detected and included intra-nuclear lipid deposition along with chromatin condensation. The results reported here demonstrate the utility of SR-FTIR and Raman spectroscopy to probe in situ DNA damage in cell nuclei resulting from UVR exposure. These results are in agreement with the increasing body of evidence that lipid accumulation is a characteristic of aggressive cancer cells, and are involved in the production of membranes for rapid cell proliferation.


Subject(s)
Cell Nucleus/radiation effects , Nucleic Acid Conformation/radiation effects , Skin/cytology , Skin/radiation effects , Cell Line, Tumor , Cell Nucleus/chemistry , DNA/chemistry , Humans , Single-Cell Analysis , Skin/chemistry , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Ultraviolet Rays
19.
J R Soc Interface ; 11(97): 20140454, 2014 Aug 06.
Article in English | MEDLINE | ID: mdl-24898023

ABSTRACT

The role that DNA conformation plays in the biochemistry of cells has been the subject of intensive research since DNA polymorphism was discovered. B-DNA has long been considered the native form of DNA in cells although alternative conformations of DNA are thought to occur transiently and along short tracts. Here, we report the first direct observation of a fully reversible en masse conformational transition between B- and A-DNA within live bacterial cells using Fourier transform infrared (FTIR) spectroscopy. This biospectroscopic technique allows for non-invasive and reagent-free examination of the holistic biochemistry of samples. For this reason, we have been able to observe the previously unknown conformational transition in all four species of bacteria investigated. Detection of this transition is evidence of a previously unexplored biological significance for A-DNA and highlights the need for new research into the role that A-DNA plays as a cellular defence mechanism and in stabilizing the DNA conformation. Such studies are pivotal in understanding the role of A-DNA in the evolutionary pathway of nucleic acids. Furthermore, this discovery demonstrates the exquisite capabilities of FTIR spectroscopy and opens the door for further investigations of cell biochemistry with this under-used technique.


Subject(s)
DNA, A-Form/chemistry , DNA, A-Form/ultrastructure , DNA, B-Form/chemistry , DNA, B-Form/ultrastructure , DNA, Bacterial/chemistry , DNA, Bacterial/ultrastructure , Desiccation , Nucleic Acid Conformation , Phase Transition , Proteus vulgaris/genetics
20.
Analyst ; 138(14): 3891-9, 2013 Jul 21.
Article in English | MEDLINE | ID: mdl-23762893

ABSTRACT

The application of FTIR spectroscopy to disease diagnosis requires a thorough knowledge of the spectroscopy associated with the cell cycle to discern disease markers from normal cellular events. We have applied synchrotron FTIR spectroscopy to monitor cells at different phases of the cell cycle namely G1, S and G2 phases. By applying Principal component analysis (PCA) from three independent trials we show clustering on a 2-dimensional scores plots (PC1 versus PC2) from cell spectra only two hours apart within the cell cycle. The corresponding PCA Loadings Plots indicate the clustering is primarily based on changes to the overall concentration of nucleic acids, proteins and lipids. During the first ten hours post mitosis, cells are observed to increase in protein and decrease in both lipid and nucleic acid concentration. During the synthesis phase, (beginning 9-11 hours post-mitosis) the PCA Loadings Plots show the accumulation of lipids within the cell as well the duplication of the genome as evidenced by strong DNA contributions. In the 4-6 hours following the synthesis phase, the cells once again accumulate protein while the relative nucleic acid and lipid concentrations decrease. These results, in comparison to previous studies on dehydrated cells, show previously unresolvable biochemical information as well as highlighting the advantages of FTIR spectroscopy applied to single living cells.


Subject(s)
Cell Cycle/physiology , Cell Physiological Phenomena , Fibroblasts/cytology , Spectroscopy, Fourier Transform Infrared/methods , Synchrotrons , Animals , Cells, Cultured , Mice , Principal Component Analysis
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