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The selective ultra-sensitive detection of a very low concentration of analyte in a liquid environment using surface-enhanced Raman spectroscopy (SERS) is a challenging task owing to the poor reproducibility of the Raman signals arising from the nonstationary nature of the substrate. However, plasmonic metal particle-incorporated microparticles can be effectively 3-D arrested in a liquid environment that can serve as a stable SERS substrate by employing an optical trapping force. Herein, we demonstrate a 3-D optically trapped Au-attached SiO2 microparticle as an efficient SERS substrate that can detect 512 pM for Rhodamine6G and 6.8 pM for crystal violet. Further, the substrate allows the simultaneous detection of multiple analytes. By utilizing the Raman signal from Rhodamine 6G as the probe beam, the selective detection of Hg2+ ions as low as 100 pM is demonstrated.
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The quenching in luminescence emission of an optically trapped ligand-free hydrophilic NaYF4:Yb, Er upconversion nanoparticle (UCNP) as a function of rose Bengal dye molecule is investigated here. The removal of oleate capping of the as-prepared UCNPs was achieved via acid treatment and characterized via FTIR and Raman spectroscopic techniques. Further, the capping removed hydrophilic single UCNP is optically trapped and the emission studies were carried out as a function of excitation laser power. Compared to the studies using the bulk solution, the single UCNP luminescence spectrum exhibited additional spectral lines. The excitation laser power-dependent studies using the bulk solution yield a slope value between 1 and 2 for Blue, Green 1, Green 2, and Red emission and thus indicate that upconversion is a two-photon upconversion process. On the other hand, in the case of laser power-dependent studies on an optically trapped single-particle study, Blue and Green 1 yield a slope value of less than 1 whereas Green 2 and Red emission gave a slope value between 1 and 2. The energy transfer studies between an optically trapped ligand-free single UCNP and the rose Bengal dye show a concentration-dependent quenching in the emission of Green emissions and illustrate the potential of developing sensor platforms.
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There has been a long-standing interest in Raman spectroscopic investigation of optically trapped single functional cells. Optical trapping using a Gaussian beam has helped researchers for decades to investigate single cells suspended in a physiological medium. However, complete and sensitive probing of single cells demands further advancements in experimental methods. Herein, we propose optical trapping and simultaneous micro-Raman spectroscopy of red blood cells (RBCs) in an unconventional face-on orientation using an optical vortex beam. Using this novel method, we are successful in comparing the conformational state of hemoglobin (Hb) molecules near the RBC membrane and inside the bulk of the cell. This method enabled us to successfully probe the oxy/deoxy ratio of Hb molecules near the RBC membrane and inside the bulk of the cell. Because of the face-on orientation, the Raman spectra of RBCs acquired using a vortex beam have a significant contribution from membrane components compared to that recorded using the Gaussian beam.
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Pinças Ópticas , Análise Espectral Raman , Membrana Celular , Eritrócitos , HemoglobinasRESUMO
Detection of urinary tract infection (UTI)-causing bacteria uses conventional time-consuming microbiological techniques. The current need is to use a fast and reliable method of bacterial identification. In order to unambiguously distinguish the UTI-causing five bacterial species used in the current study, micro-Raman spectra were obtained from a home-assembled micro-Raman system and analyzed by multivariate statistical techniques such as principal component analysis (PCA), partial least square-discriminate analysis (PLS-DA), and support vector machine (SVM). Also, the micro-Raman spectra recorded from samples containing two and three bacterial species were tested and validated against the aforementioned calibration models using PLS-DA and SVM. The prediction accuracies of up to 73 and 89% were achieved with PLS-DA and SVM, respectively. Taken together, the present study depicts the capturing of unique micro-Raman spectral features manifesting from the biochemical content of each bacterium. Also, micro-Raman spectroscopy combined with multivariate data analysis can therefore be a reliable and faster technique for the diagnosis of UTI-causing bacteria. Graphical Abstract.
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Escherichia coli/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Proteus vulgaris/isolamento & purificação , Análise Espectral Raman/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Infecções Urinárias/microbiologia , Análise Discriminante , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , Análise dos Mínimos Quadrados , Análise de Componente Principal , Infecções Estafilocócicas/diagnóstico , Máquina de Vetores de Suporte , Infecções Urinárias/diagnósticoRESUMO
Laser-induced crystallization is emerging as a promising technique to crystallize biomolecules like amino acids and proteins. The use of external materials as nucleants and novel seeding methods open new paths for protein crystallization. We report here the results of experiments that explore the effect of nucleants on laser-based crystallization of microlitre droplets of small molecules, amino acids, and proteins. The role of parameters like solute concentration, droplet volume, type and size of the nucleant, and laser power, are systematically investigated. In addition to crystallization of standard molecules like NaCl, KCl, and glycine, we demonstrate the crystallization of negatively (l-histidine), and positively (l-aspartic acid) charged amino acids and lysozyme protein. Single crystal X-ray diffraction and Raman spectroscopy studies unequivocally indicate that the nucleants do not alter the molecular structure of glycine, hydrogen bonding patterns, and packing. Localized vaporization of the solvent near the nucleant due to photothermal heating has enabled us to achieve rapid crystallization - within 3 s - at laser intensities of 0.1 MW cm-2, significantly lower than those reported earlier, with both saturated and unsaturated solutions. The outcome of the current experiments may be of utility in tackling various crystallization problems during the formation of crystals large enough to perform X-ray crystallography.
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Classification of plastics is of great importance in the recycling industry as the littering of plastic wastes increases day by day as a result of its extensive use. In this paper, we demonstrate the efficacy of a combined laser-induced breakdown spectroscopy (LIBS)-Raman system for the rapid identification and classification of post-consumer plastics. The atomic information and molecular information of polyethylene terephthalate, polyethylene, polypropylene, and polystyrene were studied using plasma emission spectra and scattered signal obtained in the LIBS and Raman technique, respectively. The collected spectral features of the samples were analyzed using statistical tools (principal component analysis, Mahalanobis distance) to categorize the plastics. The analyses of the data clearly show that elemental information and molecular information obtained from these techniques are efficient for classification of plastics. In addition, the molecular information collected via Raman spectroscopy exhibits clearly distinct features for the transparent plastics (100% discrimination), whereas the LIBS technique shows better spectral feature differences for the colored samples. The study shows that the information obtained from these complementary techniques allows the complete classification of the plastic samples, irrespective of the color or additives. This work further throws some light on the fact that the potential limitations of any of these techniques for sample identification can be overcome by the complementarity of these two techniques. Graphical Abstract á .
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A transportable reversible assembly of gold nanoparticles (AuNPs) in an aqueous environment addresses the need for in situ surafce-enhanced Raman spectroscopy (SERS) hotspot creation for biological applications. Usually, light-directed AuNP assembly methods use higher laser powers and surfactants and are, hence, unsuitable for biological applications. Here, surface plasmon polaritons-assisted dynamic assembly of AuNPs are demonstrated at laser power density as low as 100 nW µm-2 . The AuNP assembly with multiple controllable hotspots is generated in an Au-water interface for solution-based SERS measurements. The major advantage of the method is that the interparticle nanogap is tunable to achieve analyte and AuNP-specific optimum SERS enhancement. The SERS intensity is reproducible on multiple reassembly cycles and assembly attempts, proving repeatability in the produced nanogap pattern. The assembly experiments reveal the influence of AuNP surface charge and the resulting polarizability on the SPP forces. The developed system and method can detect sulforhodamine 101 (SR101) dye molecules at concentrations as low as 10-10 m. Further, the SERS measurements on double-stranded DNA suggest that the molecules are oriented in a fashion to expose adenosine to the enhanced field, leading to its dominance in the recorded spectra.
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Micro-Raman spectroscopy has emerged as one of the foremost techniques for analyzing biological cells in recent years due to its non-destructive nature and high spatial resolution. The development of optical tweezers has eased the research on biological cells as they confine living cells and organisms in the optical trap without causing much damage. Combining optical tweezers with Raman spectroscopy has opened a wide range of applications in the biomedical field as it facilitates biochemical analysis of biological samples by maintaining in-vivo conditions. Herein, we developed a light sheet-based optical tweezer that traps red blood cells (RBCs) at a very low power density spread across the whole cell, otherwise impossible with conventional optical tweezers. Furthermore, it is combined with micro-Raman spectroscopy to perform whole-cell biochemical analysis for the first time. Raman spectra of individual RBCs recorded under the line focal spot excitation are of superior quality and lack spectral signatures of photo-oxidation and heme aggregation, which is common in point focal spot excitations.
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Eritrócitos , Pinças Ópticas , Eritrócitos/química , Eritrócitos/metabolismo , Análise Espectral Raman/métodos , Heme/metabolismoRESUMO
BACKGROUND: Precise localized printing of plasmonic nanoparticles at desired locations can find a plethora of applications in diverse areas, including nanophotonics, nanomedicine, and microelectronics. The focused laser beam-assisted optical printing technique has illustrated its potential for the localized printing of differently shaped plasmonic particles. However, the technique is either time-consuming or often requires focused optical radiation, limiting its practical applications. While the optothermal printing technique has recently emerged as a promising technique for the direct and rapid printing of plasmonic nanoparticles onto transparent substrates at lower laser intensities, its potential to print the plasmonic nanoparticles to the core of the optical fiber platforms and utilize it for biological cell trapping as well as an analytical platform remains unexplored. RESULTS: Herein, we demonstrate the thermal-convection-assisted printing of the Ag plasmonic nanoparticles from the plasmonic colloidal solution onto the core of single-mode optical fiber and its multi-functional applications. The direct printing of plasmonic structure on the fiber core via the thermal-convection mechanism is devoid of the requirement of any additional chemical ligand to the fiber core. Further, we demonstrated the potential of the developed plasmonic fiber probe as a multifunctional surface-enhanced Raman spectroscopic (SERS) platform for sensing, chemical reaction monitoring, and single-cell studies. The developed SERS fiber probe is found to detect crystal violet in an aqueous solution as low as 100 pM, with a plasmonic enhancement of 107. Additionally, the capability of the fiber-tip platform to monitor the surface plasmon-driven chemical reaction of 4-nitrothiophenol (4NTP) dimerizing into p, p'-dimercaptoazobenzene (DMAB) is demonstrated. Further, the versatility of the fiber probe as an effective platform for opto-thermophoretic trapping of single biological cells such as yeast, along with its Raman spectroscopic studies, is also shown here. SIGNIFICANCE: In this study, we illustrate for the first time the optothermal direct printing of plasmonic nanoparticles onto the core of a single-mode fiber. Further, the study demonstrates that such plasmonic nanoparticle printed fiber tip can act as a multi-functional analytical platform for optothermally trap biological particles as well as monitoring plasmon-driven chemical reactions. In addition, the plasmonic fiber tip can be used as a cost-effective SERS analytical platform and is thus expected to find applications in diverse areas.
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Nanopartículas Metálicas , Fibras Ópticas , Prata , Análise de Célula Única , Análise Espectral Raman , Análise Espectral Raman/métodos , Nanopartículas Metálicas/química , Prata/química , Compostos de Sulfidrila/química , Fenóis/análise , Fenóis/química , Humanos , ImpressãoRESUMO
BACKGROUND: Plastic Solid Waste (PSW) sorting is a procedure of paramount importance in the mechanical recycling process of plastics waste. The major limitation of the techniques relying on physical properties of plastics is the time taken for analysis and poor accuracy. Spectroscopy has been shown to be a suitable method in plastic sorting due to its atomic and molecular characterization capabilities, and ability to give results in very short time scales. However, for practical applications it is essential to translate this technique into an automatic technology, by combining it with advanced chemometric tools which can give observer independent judgement. RESULTS: The indigenously developed bi-model Laser Induced Breakdown Spectroscopy (LIBS)-Raman system with single source and single detector can record the LIBS-Raman spectral signals in single-shot mode in a total time frame of 20 ms. Out of the combinations of Principal Component Analysis (PCA) and Partial Least Squares (PLS) with Logistic Regression (LR), Linear Discriminant Analysis (LDA), Support Vector Machine (SVM), and Partial Least Squares-Discriminant Analysis (PLS-DA) based classifiers, the PLS-DA based model showed the maximum classification accuracy with 95 % based on LIBS data and 100 % based on Raman data. The reliability of the model was assessed using 4-fold cross-validation which showed a sensitivity of 90.28 % and specificity of 98.29 % for predictions based on LIBS data, and 99 % sensitivity and 99.82 % specificity for predictions relying on Raman data. SIGNIFICANCE: The results show how the combination of multimodal spectroscopy with chemometric analysis enhances the applicability of spectroscopic techniques for plastic sorting. The classification model successfully classified seven types of post-consumer plastic samples based on combined LIBS and Raman data. With the home-built software for automated prediction, the system takes less than a second to predict the plastic type illustrating the potential of the method for translation to regular routine industrial applications.
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BACKGROUND: The thermally coupled energy states that contribute to the upconversion luminescence of rare earth element-doped nanoparticles have been the subject of intense research due to their potential nanoscale temperature probing. However, the inherent low quantum efficiency of these particles often limits their practical applications, and currently, surface passivation and incorporation of plasmonic particles are being explored to improve the inherent quantum efficiency of the particle. However, the role of these surface passivating layers and the attached plasmonic particles in the temperature sensitivity of upconverting nanoparticles while probing the intercellular temperature has not been investigated thus far, particularly at the single nanoparticle level. RESULTS: The analysis of the study on the thermal sensitivity of oleate-free UCNP, UCNP@SiO2, and UCNP@SiO2@Au particles is carried out at a single particle level in a physiologically relevant temperature range (299 K-319 K) by optically trapping the particle. The thermal relative sensitivity of the as-prepared upconversion nanoparticle (UCNP) is found to be greater than that of UCNP@SiO2 and UCNP@SiO2@Au particles in an aqueous medium. An optically trapped single luminescence particle inside the cell is used to monitor the temperature inside the cell by measuring the luminescence from the thermally coupled states. The absolute sensitivity of optically trapped particles inside the biological cell increases with temperature, with a greater impact on the bare UCNP, which exhibits higher values for thermal sensitivity than UCNP@SiO2 and UCNP@SiO2@Au. The thermal sensitivity of the trapped particle inside the biological cell at 317 K indicates the thermal sensitivity of UCNP > UCNP@SiO2@Au > UCNP@SiO2 particles. SIGNIFICANCE AND NOVELTY: Compared to bulk sample-based temperature probing, the present study demonstrates temperature measurement at the single particle level by optically trapping the particle and further explores the role of the passivating silica shell and the incorporation of plasmonic particles on thermal sensitivity. Furthermore, thermal sensitivity measurements inside a biological cell at the single particle level are investigated and illustrated that thermal sensitivity at a single particle is sensitive to the measuring environment.
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Ever-accumulating amounts of plastic waste raises alarming concern over environmental and public health. A practical solution for addressing this threat is recycling, and the success of an industry-oriented plastic recycling system relies greatly on the accuracy of the waste sorting technique adapted. We propose a multi-modal spectroscopic sensor which combines laser-induced breakdown spectroscopy (LIBS) and Raman spectroscopy in a single optical platform for characterizing plastics based on elemental and molecular information to assist the plastic identification-sorting process in recycling industries. The unique geometry of the system makes it compact and cost-effective for dual spectroscopy. The performance of the system in classifying industrially important plastic classes counting PP, PC, PLA, Nylon-1 1, and PMMA is evaluated, followed by the application of the same in real-world plastics comprising PET, HDPE, and PP in different chemical-physical conditions where the system consumes less than 30 ms for acquiring LIBS-Raman signals. The evaluation of the system in characterizing commuting samples shows promising results to be applied in industrial conditions in future. The study on effect of physical-chemical conditions of plastic wastes in characterizing them using the system shows the necessity for combining multiple techniques together. The proposal is not to determine the paramount methodology to characterize and sort plastics, but to demonstrate the advantages of dual-spectroscopy sensors in such applications. The outcomes of the study suggest that the system developed herein has the potential of emerging as an industrial-level plastic waste sorting sensor.
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Plásticos , Gerenciamento de Resíduos , Resíduos Industriais , Indústrias , Reciclagem , Análise Espectral RamanRESUMO
Malaria, dengue and leptospirosis are three tropical infectious diseases that present with severe hematological derangement causing significant morbidity and mortality, especially during the seasonal monsoons. During the course of these infectious diseases, circulating red blood cells are imperiled to the direct ill-effects of the infectious pathogen in the body as well as to the pro-inflammatory cytokines generated as a consequence of the infection. RBCs when exposed to such inflammatory and/or pathogenic milieu are susceptible to injuries such as RBC programmed eryptosis or RBC programmed necrosis. This research aimed to explore the Raman spectra of live red cells that were extracted from patients infected with malaria, dengue, and leptospirosis. Red cells were optically trapped and micro-Raman probed using a 785 nm Diode laser. RBCs from samples of all three diseases displayed Raman signatures that were significantly altered from the normal/healthy. Distinct spectral markers that were common across all the four groups were obtained from various standardized multivariate analytical methods. Following comprehensive examination of multiple studies, we propose these spectral wavenumbers as "Raman markers of RBC injury." Findings in our study display that anemia-triggering infections can inflict variations in the healthy status of red cells, easily identifiable by selectively analyzing specific Raman markers. Additionally, this study also highlights relevant statistical tools that can be utilized to study Raman spectral data from biological samples which could help identify the very significant Raman peaks from the spectral band. This approach of RBC analysis can foster a better understanding of red cell behavior and their alterations exhibited in health and disease.
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3-Benzylchroman-4-ones (homoisoflavanones) are oxygen-containing heterocycles with a sixteen-carbon skeleton. They belong to the class of naturally occurring polyphenolic flavonoids with limited occurrence in nature and possess anti-inflammatory, antibacterial, antihistaminic, antimutagenic, antiviral, and angioprotective properties. Recently, we reported the synthesis and anticancer activity studies of fifteen 3-benzylchroman-4-one molecules, and most of them were proven to be effective against BT549 and HeLa cells. In this work, we report the single-crystal X-ray crystallographic studies of two molecules 3-[(2-hydroxyphenyl)methyl]-3,4-dihydro-2H-1-benzopyran-4-one and 3-[(2,4-dimethoxyphenyl)methyl]-3,4-dihydro-2H-1-benzopyran-4-one. The single crystals were grown using a novel laser-induced crystallization technique. We observed that the 3-benzylchroman-4-one derivative bearing OH substitution at the 2' position adopted different conformation due to formation of dimers through O-Hâ¯O, and C-Hâ¯O intermolecular hydrogen bondings. The role of OH substitution in the aforementioned conformational changes was evaluated using density functional theory (DFT), Hirshfeld surface, energy framework and FTIR spectroscopy analysis. In addition, we have carried out a Cambridge Structural Database (CSD) study to understand the conformational changes using five analogue structures. X-ray crystallographic, computational, and spectroscopic studies of 3-benzylchroman-4-ones provided an insight into the role of substitution at benzyl moieties in stabilizing the three-dimensional (3D) structures.
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Introduction: The unprecedented outbreaks of corona virus disease of 2019 (COVID-19) have highlighted the necessity of readily available, reliable, precise, and faster techniques for its detection. Nasopharyngeal swab has been the gold standard for the diagnosis of COVID-19. However, it is not an ideal screening procedure for massive screening as it implicates the patient's stay in the hospital or at home until diagnosis, thus causing crowding of the specimen at the diagnostic centers. Present study deal with the exploration of potential application of different body fluids using certain highly objective techniques (Optical and e-Nose) for faster detection of molecular markers thereby diagnosing viral infections.Areas covered: This report presents an evaluation of different body fluids, and their advantages for the rapid detection of COVID-19, coupled with highly sensitive optical techniques for the detection of molecular biomarkers.Expert opinion: Tears, saliva, and breath samples can provide valuable information about viral infections. Our brief review strongly recommends the application of saliva/tears and exhaled breath as clinical samples using technics such as high-performance liquid chromatography-laser-induced fluorescence, photoacoustic spectroscopy, and e-Nose, respectively, for the fast diagnosis of viral infections.
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Teste para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Biomarcadores/metabolismo , Líquidos Corporais/virologia , Testes Respiratórios , Cromatografia Líquida , Expiração , Humanos , Lasers , Programas de Rastreamento/métodos , Nanotecnologia , Técnicas Fotoacústicas , Saliva/virologia , Sensibilidade e Especificidade , Lágrimas/virologiaRESUMO
The influence of static electric field of about 4.9â¯kV/cm on optically trapped polystyrene bead has been studied using micro-Raman spectroscopy. This experimental method could explore the response of electronic polarizability of molecules to the static electric field by measuring the Raman intensity. It is observed that, the atomic electrons in polystyrene will polarize to the extent that they induce a Raman intensity enhancements by a factor ranging from 0.004 to 0.64 for various spectral bands of polystyrene when exposed to the electric field from ~4.90 to 4.96â¯kV/cm.
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Derangements in bilirubin metabolism and/or dysfunctions in the hepato-biliary system lead to the unhealthy buildup of bilirubin in blood, resulting in jaundice. During the course of this disorder, circulating red cells are invariably subjected to toxic effects of serum bilirubin and an array of inflammatory compounds. This study aimed to investigate the vibrational spectroscopy of live red cells in jaundice using micro-Raman spectroscopy combined with optical-trap. Red cells from blood samples of healthy volunteers and patients with jaundice were optically immobilized and micro-Raman probed using a 785 nm diode laser. Raman signatures from red cells in jaundice exhibited significant variations from the normal and the spectral-markers were obtained from multivariate analytical methods. This research gives insightful views on how different pathologies can act as "stress-milieus" for red cells in circulation, possibly impeding their normal functions and also exasperating anemia. Raman spectroscopy, an emerging bio-analytical technique, is sensitive in detecting molecular-conformations in situ, at cellular-levels and in real-time. This study could pave way in understanding fundamental red cell behavior in different diseases by analyzing Raman markers.
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Laser-induced crystallization is emerging as an alternative technique to crystallize biomolecules. However, its applications are limited to specific small molecules and some simple proteins, possibly because of the need to use high-intensity, pulsed lasers and relatively long laser irradiation time. Both these factors tend to denature biological molecules. If the laser-intensity and time required to crystallize biomolecules were to be reduced, laser-induced crystallization may well become of widespread utility. We report here the crystallization of nineteen natural amino acids by a laser-induced method in combination with one of three nucleants: aluminum, coconut coir, and peacock feather barbule. We have utilized a low-power, continuous wave (CW) Nd:YAG laser (λ = 1064 nm). The advantages of our method are (i) the use of very small laser powers (60 mW), and (ii) the ability to obtain diffraction quality crystals within a mere few seconds. For most amino acids our method yields several orders of magnitude reduction in crystallization time. The use of biocompatible nucleants like coir fibres and peacock feather barbules are novel; their non-toxic nature may find broad applicability in rapid crystallization of diverse biological molecules.
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Aminoácidos/química , Materiais Biocompatíveis/química , Cristalização , Cristalização/métodos , Lasers de Estado Sólido , Microscopia Eletrônica de Varredura , SolubilidadeRESUMO
We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of ß-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to ß-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of ß-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells.
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Eritrócitos/efeitos dos fármacos , Raios Infravermelhos , beta Caroteno/farmacologia , Plaquetas/química , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Eritrócitos/química , Eritrócitos/citologia , Eritrócitos/efeitos da radiação , Humanos , Linfócitos/química , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Análise Espectral RamanRESUMO
Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.