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Optical Photothermal Infrared (O-PTIR) spectroscopy is a new technique for measuring submicron spatial resolution IR spectra with little or no sample preparation. This speeds up analysis times benefiting high-volume manufacturers through gaining insight into process contamination that occurs during development and on production lines. The ability to rapidly obtain far-field non-contact IR spectra at high spatial resolution facilitates the chemical identification of small organic contaminants that are not possible to measure with conventional Fourier transform infrared (FT-IR) microspectroscopy. The unique pump-probe system architecture also facilitates submicron simultaneous IR + Raman microscopy from the same spot with the same spatial resolution. With these unique capabilities, O-PTIR is finding utilization in the high-volume and high-value industries of high-tech componentry (memory storage, electronics, displays, etc.).
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Optical photothermal infrared (O-PTIR) and Raman spectroscopy and imaging was used to explore the spatial distributions of molecular constituents of a laminate sample consisting of the bioplastics, polyhydroxyalkanoate (PHA) and polylactic acid (PLA), near the interfacial boundary. Highly spatially resolved simultaneous IR and Raman spectra were sequentially collected at 100 nm increments along a line traversing the interface. The set of spectra were subjected to 2D-COS analysis to extract the detailed nature of the spatial distribution of the laminate constituents. It was revealed that the laminate is not a simple binary system of two non-interacting polymers, but consists of different constituents with more complex spatial distributions. Some portion of PLA seems to penetrate into the PHA layer. The crystallinity of PHA near the interface is reduced compared to the rest of the PHA layer. The result suggests the existence of some partial molecular mixing even for these seemingly immiscible polymer pairs. The mixing probably occurs at the segmental level confined to only several hundred nanometers of space at the interface. Such partial mixing may explain the high compatibility between the two bioplastics.
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During their lifespan, Red blood cells (RBC), due to their inability to self-replicate, undergo an ageing degradation phenomenon. This pathway, both in vitro and in vivo, consists of a series of chemical and morphological modifications, which include deviation from the biconcave cellular shape, oxidative stress, membrane peroxidation, lipid content decrease and uncoupling of the membrane-skeleton from the lipid bilayer. Here, we use the capabilities of atomic force microscopy based infrared nanospectroscopy (AFM-IR) to study and correlate, with nanoscale resolution, the morphological and chemical modifications that occur during the natural degradation of RBCs at the subcellular level. By using the tip of an AFM to detect the photothermal expansion of RBCs, it is possible to obtain nearly two orders of magnitude higher spatial resolution IR spectra, and absorbance images than can be obtained on diffraction-limited commercial Fourier-transform Infrared (FT-IR) microscopes. Using this approach, we demonstrate that we can identify localized sites of oxidative stress and membrane peroxidation on individual RBC, before the occurrence of neat morphological changes in the cellular shape.
Assuntos
Eritrócitos/citologia , Microscopia de Força Atômica/métodos , Estresse Oxidativo , Espectrofotometria Infravermelho/métodos , Forma Celular , Contagem de Eritrócitos , Eritrócitos/química , Humanos , Peroxidação de Lipídeos , Lipídeos de Membrana/química , NanotecnologiaRESUMO
Poly[(R)-3-hydroxyalkanoate]s or PHAs are aliphatic polyesters produced by numerous microorganisms. They are accumulated as energy and carbon reserve in the form of small intracellular vesicles. Poly[(R)-3-hydroxybutyrate] (PHB) is the most ubiquitous and simplest PHA. An atomic force microscope coupled with a tunable infrared laser (AFM-IR) was used to record highly spatially resolved infrared spectra of commercial purified PHB and native PHB within bacteria. For the first time, the crystallinity degree of native PHB within vesicle has been directly evaluated in situ without alteration due to the measure or extraction and purification steps of the polymer: native PHB is in crystalline state at 15% whereas crystallinity degree reaches 57% in commercial PHB. Chloroform addition on native PHB induces crystallization of the polymer within bacteria up to 60%. This possibility of probing and changing the physical state of polymer in situ could open alternative ways of production for PHB and others biopolymers. Graphical abstract An atomic force microscope coupled with a tunable infrared laser (AFM-IR) has been used to record local infrared spectra of biopolymer PHB within bacteria. Deconvolution of those spectra has allowed to determine in situ the crystallinity degree of native PHB.
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Clorofórmio/farmacologia , Hidroxibutiratos/química , Polímeros/química , Rhodobacter sphaeroides/efeitos dos fármacos , Varredura Diferencial de Calorimetria , Cristalização , Microscopia de Força Atômica , Pós , Rhodobacter sphaeroides/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The toxicity of nanomaterials raises major concerns because of the impact that nanomaterials may have on health, which remains poorly understood. We need to explore the fate of individual nanoparticles in cells at nano and molecular levels to establish their safety. Conformational changes in secondary protein structures are one of the main indicators of impaired biological function, and hence, the ability to identify these changes at a nanoscale level offers unique insights into the nanotoxicity of materials. Here, we used nanoscale infrared spectroscopy and demonstrated for the first time that nanodiamond-induced alterations in both extra- and intracellular secondary protein structures lead to the formation of antiparallel ß-sheet, ß-turns, intermolecular ß-sheet, and aggregation of proteins. These conformational changes of the protein structure may result in the loss of functionality of proteins and in turn lead to adverse effects.
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Nanodiamantes/química , Proteínas/efeitos dos fármacos , Animais , Fibronectinas/química , Fibronectinas/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Microscopia de Força Atômica , Conformação Proteica em Folha beta/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas/química , Ratos , Espectrofotometria Infravermelho , Esferoides Celulares/química , Esferoides Celulares/efeitos dos fármacosRESUMO
Bone has a hierarchical structure extending from the micrometer to the nanometer scale. We report here the first analysis of non-human primate osteonal bone obtained using a spectrometer coupled to an AFM microscope (AFM-IR), with a resolution of 50-100 nm. Average spectra correspond to those observed with conventional FTIR spectroscopy. The following validated FTIR parameters were calculated based on intensities observed in scans covering ~60 µm from the osteon center: mineral content (1030/1660 cm(-1)), crystallinity (1030/1020 cm(-1)), collagen maturity (1660/1690 cm(-1)), and acid phosphate content (1128/1096 cm(-1)). A repeating pattern was found in most of these calculated IR parameters corresponding to the reported inter- and intra-lamellar spacing in human bone, indicating that AFM-IR measurements will be able to provide novel compositional information on the variation in bone at the nanometer level.
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Osso e Ossos/química , Osso e Ossos/ultraestrutura , Animais , Microscopia de Força Atômica , Papio , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The recent combination of atomic force microscopy and infrared spectroscopy (AFM-IR) has led to the ability to obtain IR spectra with nanoscale spatial resolution, nearly two orders-of-magnitude better than conventional Fourier transform infrared (FT-IR) microspectroscopy. This advanced methodology can lead to significantly sharper spectral features than are typically seen in conventional IR spectra of inhomogeneous materials, where a wider range of molecular environments are coaveraged by the larger sample cross section being probed. In this work, two-dimensional (2D) correlation analysis is used to examine position sensitive spectral variations in datasets of closely spaced AFM-IR spectra. This analysis can reveal new key insights, providing a better understanding of the new spectral information that was previously hidden under broader overlapped spectral features. Two examples of the utility of this new approach are presented. Two-dimensional correlation analysis of a set of AFM-IR spectra were collected at 200-nm increments along a line through a nucleation site generated by remelting a small spot on a thin film of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). There are two different crystalline carbonyl band components near 1720 cm-1 that sequentially disappear before a band at 1740 cm-1 due to more disordered material appears. In the second example, 2D correlation analysis of a series of AFM-IR spectra spaced every 1 micrometer of a thin cross section of a bone sample measured outward from an osteon center of bone growth. There are many changes in the amide I and phosphate band contours, suggesting changes in the bone structure are occurring as the bone matures.
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Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument (AFM-IR) capable of producing IR spectra and absorption images at a sub-micrometer spatial resolution. This new device enables human hair to be spectroscopically characterized at levels not previously possible. In particular, it was possible to determine the location of structural lipids in the cuticle and cortex of hair. Samples of human hair were embedded, cross-sectioned, and mounted on ZnSe prisms. A tunable IR laser generating pulses of the order of 10 ns was used to excite sample films. Short duration thermomechanical waves, due to infrared absorption and resulting thermal expansion, were studied by monitoring the resulting excitation of the contact resonance modes of the AFM cantilever. Differences are observed in the IR absorbance intensity of long-chain methylene-containing functional groups between the outer cuticle, middle cortex, and inner medulla of the hair. An accumulation of structural lipids is clearly observed at the individual cuticle layer boundaries. This method should prove useful in the future for understanding the penetration mechanism of substances into hair as well as elucidating the chemical nature of alteration or possible damage according to depth and hair morphology.
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Cabelo/química , Cabelo/ultraestrutura , Lipídeos/análise , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Espectrofotometria Infravermelho/métodos , Desenho de Equipamento , Preparações para Cabelo/química , Preparações para Cabelo/farmacocinética , Humanos , Queratinas/química , Queratinas/ultraestrutura , Lasers , Microscopia de Força Atômica/instrumentação , Nanotecnologia/instrumentação , Manejo de Espécimes , Espectrofotometria Infravermelho/instrumentação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , VibraçãoRESUMO
An atomic force microscope (AFM) and a tunable infrared (IR) laser source have been combined in a single instrument (AFM-IR) capable of producing ~200-nm spatial resolution IR spectra and absorption images. This new capability enables IR spectroscopic characterization of human stratum corneum at unprecendented levels. Samples of normal and delipidized stratum corneum were embedded, cross-sectioned and mounted on ZnSe prisms. A pulsed tunable IR laser source produces thermomechanical expansion upon absorption, which is detected through excitation of contact resonance modes in the AFM cantilever. In addition to reducing the total lipid content, the delipidization process damages the stratum corneum morphological structure. The delipidized stratum corneum shows substantially less long-chain CH2 -stretching IR absorption band intensity than normal skin. AFM-IR images that compare absorbances at 2930/cm (lipid) and 3290/cm (keratin) suggest that regions of higher lipid concentration are located at the perimeter of corneocytes in the normal stratum corneum.
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Epiderme/metabolismo , Lipídeos/química , Microscopia de Força Atômica/métodos , Espectrofotometria Infravermelho/métodos , Absorção , Epiderme/ultraestrutura , Desenho de Equipamento , Humanos , Queratinas/química , Lasers , Compostos de Selênio/química , Compostos de Zinco/químicaRESUMO
Polymer and life science applications of a technique that combines atomic force microscopy (AFM) and infrared (IR) spectroscopy to obtain nanoscale IR spectra and images are reviewed. The AFM-IR spectra generated from this technique contain the same information with respect to molecular structure as conventional IR spectroscopy measurements, allowing significant leverage of existing expertise in IR spectroscopy. The AFM-IR technique can be used to acquire IR absorption spectra and absorption images with spatial resolution on the 50 to 100 nm scale, versus the scale of many micrometers or more for conventional IR spectroscopy. In the life sciences, experiments have demonstrated the capacity to perform chemical spectroscopy at the sub-cellular level. Specifically, the AFM-IR technique provides a label-free method for mapping IR-absorbing species in biological materials. On the polymer side, AFM-IR was used to map the IR absorption properties of polymer blends, multilayer films, thin films for active devices such as organic photovoltaics, microdomains in a semicrystalline polyhydroxyalkanoate copolymer, as well as model pharmaceutical blend systems. The ability to obtain spatially resolved IR spectra as well as high-resolution chemical images collected at specific IR wavenumbers was demonstrated. Complementary measurements mapping variations in sample stiffness were also obtained by tracking changes in the cantilever contact resonance frequency. Finally, it was shown that by taking advantage of the ability to arbitrarily control the polarization direction of the IR excitation laser, it is possible to obtain important information regarding molecular orientation in electrospun nanofibers.
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Determining the extent of miscibility of amorphous components is of great importance for certain pharmaceutical systems, in particular for polymer-polymer and polymer-small molecule blends. In this study, the application of standard atomic force microscopy (AFM) measurements combined with nanoscale mid-infrared (mid-IR) spectroscopy was explored to evaluate miscibility in binary polymer blends. The miscibility characteristics of a set of 50/50 (w/w) polymer blends comprising of poly(vinylpyrrolidone) (PVP) with dextran or maltodextrin (DEX) of varying molecular weights (MWs) were investigated. Standard AFM characterization results show good agreement with inferences drawn from differential scanning calorimetry (DSC) analysis in terms of forming either single or two phase systems. AFM analysis also provided insight into the microstructure of the two phase systems and how domain sizes varied as a function of polymer MWs. Nanoscale mid-IR evaluation of the blends, performed by collecting local mid-IR spectra or spectral maps, provided an extra dimension of information about the dependence of polymer MWs on chemical composition of the different phases. AFM, combined with nanoscale mid-infrared analysis, thus appears to be a promising technique for the evaluation of miscibility in certain pharmaceutical blends.
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Dextranos/química , Polímeros/química , Polissacarídeos/química , Povidona/química , Microscopia de Força AtômicaRESUMO
The applicability of nanoscale mid-infrared (mid-IR) spectroscopy for the study of the micro- and nanostructure of pharmaceutical drug-polymer systems was explored. Felodipine-poly(acrylic acid) (PAA) blends were used as model systems. Standard atomic force microscopy evaluation as a function of drug-polymer composition suggested limited miscibility, in line with previous findings. Localized spectra on a 50:50 (w/w) felodipine-PAA dispersion revealed that the discrete submicrometer domains formed corresponded to an amorphous felodipine-rich phase while the continuous phase tended to be rich in PAA. Further, spectroscopic imaging at selected wavenumbers, enabling discrimination between both constituents, confirmed this finding and made it possible to chemically image differences in composition between each phase with submicrometer resolution.
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Nanotecnologia , Preparações Farmacêuticas/química , Polímeros/química , Microscopia de Força Atômica , Espectrofotometria InfravermelhoRESUMO
Several prism-based spectrographs employing a mercury cadmium telluride (MCT) focal plane array detector have been interfaced to an infrared microscope. In the combined system, the area-defining aperture of the microscope also served as the entrance slit to the spectrograph. This investigation considered the fundamental limits of diffraction for both the spectrograph and microscope in order to determine both the spatial and spectral resolution of the system as a whole. Experimental results for spectral resolution, spectral range, and peak-to-peak noise have been presented. Finally, the dynamic capabilities of one spectrograph/microscope combination were investigated.
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Espectrofotometria Infravermelho/instrumentação , Espectrofotometria Infravermelho/métodos , Compostos de Cádmio/química , Fluoreto de Cálcio/química , Compostos de Mercúrio/química , Polímeros/química , Cloreto de Sódio/química , Xilenos/químicaRESUMO
Atomic force microscopy (AFM) and infrared (IR) spectroscopy have been combined in a single instrument (AFM-IR) capable of producing sub-micrometer spatial resolution IR spectra and absorption images. This new capability enables the spectroscopic characterization of microdomain-forming polymers at levels not previously possible. Films of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were solution cast on ZnSe prisms, followed by melting and annealing to generate crystalline microdomains of different sizes. A tunable IR laser generating pulses of the order of 10 ns was used for excitation of the sample films. Short duration thermomechanical waves, due to infrared absorption and resulting thermal expansion, were studied by monitoring the resulting excitation of the contact resonance modes of the AFM cantilever. Dramatic differences in the room-temperature IR spectra are observed in the 1200-1300 cm(-1) range as a function of position on a spatial scale of less than one micrometer. This spectral region is particularly sensitive to the polymer backbone conformation. Such dramatic spectral differences have also been observed previously in bulk IR measurements, but only by comparing room-temperature spectra with ones collected at higher temperatures. Less dramatic, but significant, AFM-IR spectral differences are observed in the carbonyl stretching region around 1720 cm(-1) as a function of location on the sample. Two overlapping, but relatively sharp, carbonyl bands are observed near 1720 cm(-1) in more crystalline regions of the polymer, while a broader carbonyl stretching band appears centered at 1740 cm(-1) in the more amorphous regions. Using this spectral region, it is possible to monitor the development of polymer crystalline structures at varying distances from a nucleation site, where the site was generated by bringing a heated AFM tip close to a specific location to locally anneal the sample.
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Advantages and limitations of analyzing authentic and counterfeit pharmaceutical tablets with both macro (nonimaging) attenuated total internal reflection Fourier transform infrared (ATR-FT-IR) spectroscopy and micro ATR-FT-IR spectroscopic imaging have been evaluated. The results of this study demonstrated that micro ATR imaging was more effective for extracting formulation information (sourcing), whereas a macro ATR approach was better suited for counterfeit detection (screening). More importantly, this study demonstrated that a thorough analysis of the counterfeit core can be achieved by combining the results of both techniques.
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Medicamentos Falsificados/análise , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Comprimidos/química , Modelos TeóricosAssuntos
Espectrofotometria Infravermelho/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Detergentes/química , Desenho de Equipamento , Polímeros/química , Espectrofotometria Infravermelho/instrumentação , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Estresse Mecânico , TemperaturaRESUMO
Previously, we established the ability to collect infrared microspectroscopic images of large areas using a large radius hemisphere internal reflection element (IRE) with both a single point and a linear array detector. In this paper, preliminary work in applying this same method to a focal plane array (FPA) infrared imaging system is demonstrated. Mosaic tile imaging using a large radius germanium hemispherical IRE on a FPA Fourier transform infrared microscope imaging system can be used to image samples nearly 1.5 mm x 2 mm in size. A polymer film with a metal mask is imaged using this method for comparison to previous work. Images of hair and skin samples are presented, highlighting the complexity of this method. Comparisons are made between the linear array and FPA methods.
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Ciências Forenses/instrumentação , Germânio , Cabelo/química , Polietilenos/química , Pele/química , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Ciências Forenses/métodos , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
A comparison of prism-based spectrographs to grating-based spectrographs is made when each of the systems is coupled to a modern-day liquid-nitrogen-cooled photovoltaic array detector. A comparison of the systems is also made using a room-temperature microbolometer array detector. Finally, infrared microspectroscopy of samples whose size is approximately 10 micrometers will be demonstrated using a prism spectrograph outfitted with both types of detectors. The results of the study show that prism-based spectrographs offer an economical alternative to grating-based systems when spectral coverage is more critical than spectral resolution. The results also demonstrate that spectra with good signal-to-noise ratios can be collected on any of the systems with a total integration time of 10 seconds or less.
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Refratometria/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Espectrofotometria Infravermelho/instrumentação , Transdutores , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Properties of polymer alloys comprising poly(lactic acid) and Nodax copolymers are investigated. Nodax is a family of bacterially produced polyhydroxyalkanoate (PHA) copolymers comprising 3-hydroxybutyrate (3HB) and other 3-hydroxyalkanoate (3HA) units with side groups greater than or equal to three carbon units. The incorporation of 3HA units with medium-chain-length (mcl) side groups effectively lowers the crystallinity and the melt temperature, Tm, of this class of PHA copolymers, in a manner similar to that of alpha olefins controlling the properties of linear low density polyethylene. The lower Tm makes the material easier to process, as the thermal decomposition temperature of PHAs is then relatively low. The reduced crystallinity provides the ductility and toughness required for many plastics applications. When a small amount of ductile PHA is blended with poly(lactic acid) (PLA), a new type of polymer alloy with much improved properties is created. The toughness of PLA is substantially increased without a reduction in the optical clarity of the blend. The synergy between the two materials, both produced from renewable resources, is attributed to the retardation of crystallization of PHA copolymers finely dispersed in a PLA matrix as discrete domains.