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1.
Nat Commun ; 10(1): 4764, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628307

RESUMO

Water is arguably the most common and yet least understood material on Earth. Indeed, the biophysical behavior of water in crowded intracellular milieu is a long-debated issue. Understanding of the spatial and compositional heterogeneity of water inside cells remains elusive, largely due to a lack of proper water-sensing tools with high sensitivity and spatial resolution. Recently, stimulated Raman excited fluorescence (SREF) microscopy was reported as the most sensitive vibrational imaging in the optical far field. Herein we develop SREF into a water-sensing tool by coupling it with vibrational solvatochromism. This technique allows us to directly visualize spatially-resolved distribution of water states inside single mammalian cells. Qualitatively, our result supports the concept of biological water and reveals intracellular water heterogeneity between nucleus and cytoplasm. Quantitatively, we unveil a compositional map of the water pool inside living cells. Hence we hope SREF will be a promising tool to study intracellular water and its relationship with cellular activities.


Assuntos
Microscopia de Fluorescência/métodos , Microscopia Óptica não Linear/métodos , Análise de Célula Única/métodos , Água/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Fenômenos Fisiológicos Celulares , Cor , Citoplasma/química , Citoplasma/metabolismo , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Reprodutibilidade dos Testes , Solventes/química , Vibração , Água/química
2.
Nat Methods ; 16(9): 830-842, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471618

RESUMO

All molecules consist of chemical bonds, and much can be learned from mapping the spatiotemporal dynamics of these bonds. Since its invention a decade ago, stimulated Raman scattering (SRS) microscopy has become a powerful modality for imaging chemical bonds with high sensitivity, resolution, speed and specificity. We introduce the fundamentals of SRS microscopy and review innovations in SRS microscopes and imaging probes. We highlight examples of exciting biological applications, and share our vision for potential future breakthroughs for this technology.


Assuntos
Substâncias Macromoleculares/análise , Imagem Molecular/métodos , Microscopia Óptica não Linear/métodos , Animais , Humanos
3.
Appl Microbiol Biotechnol ; 103(16): 6759-6769, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31230100

RESUMO

Confocal Raman microspectral imaging (CRMI) is an advanced cell-imaging method that maps endogenous molecular compositions with their unique spectral fingerprint indicators. The aim of this work was to provide a visualized understanding of subcellular features of live osteosarcoma cells using a 532-nm laser excitation without the use of dyes or molecular probes. Both malignant osteoblast and spindle osteosarcoma cells derived from the BALB/c mouse osteosarcoma cell line K7M2 were investigated in this work. After preprocessing the obtained spectral dataset, K-means cluster analysis (KCA) is employed to reconstruct Raman spectroscopic maps of single biological cells by identifying regions of the cellular membrane, cytoplasm, organelles, and nucleus with their corresponding mean spectra. Principal component analysis (PCA) was further employed to indicate variables of significant influence on the separation of the spectra of each cellular component. The biochemical components of the two cell types were then extracted by showing the spectral and distribution features attributed to proteins, lipids, and DNA. Using this standardized CRMI technique and multivariate analysis approaches, the results obtained could be a sound foundation for a typical Raman imaging protocol of live cellular biomedical analysis.


Assuntos
Fatores Biológicos/análise , Linhagem Celular Tumoral/química , Microscopia Óptica não Linear/métodos , Osteossarcoma/patologia , Análise de Célula Única/métodos , Animais , Camundongos , Camundongos Endogâmicos BALB C , Análise Multivariada
4.
PLoS One ; 14(5): e0216811, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112567

RESUMO

Lipid droplets are lipid-storage organelles with a key role in lipid accumulation pathologies such as diabetes, obesity and atherosclerosis. Despite their important functions many aspects of lipid droplets biology are still unknown. This is partially due to the current use of exogenous labels to monitor their formation and remodelling by invasive imaging methods. Here, we apply stimulated Raman scattering microscopy to acquire images with high spatial resolution along with resolving capabilities of lipids and proteins and three-dimensional sectioning. Our images and data analysis demonstrate an increase in the number of large (>15µm2) lipid droplets in human adipocyte cells during differentiation process. In addition, spatially-resolved maps of lipids and proteins inside cells and three dimensional reconstructions of lipids at the initial and final steps of adipocyte differentiation are reported, too.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Imagem Tridimensional , Gotículas Lipídicas/metabolismo , Microscopia Óptica não Linear , Células 3T3-L1 , Animais , Camundongos
6.
Theranostics ; 9(5): 1348-1357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867835

RESUMO

Antibiotics resistance developed by biofilms has posed a clinical challenge in the effective treatment of bacterial infections. However, the resistance mechanisms have not been well understood due to a lack of suitable tools for dynamic observation of the interplay between antibiotics and biofilm. In this work, with the use of rapid hyperspectral stimulated Raman scattering microscopy associated with an aryl-alkyne-based Raman tag synthesized, we investigate dynamic interactions between vancomycin and Staphylococcus aureus (S. aureus) biofilm to gain new insights into the resistance mechanisms of the biofilm. Methods: We utilize spectral focusing hyperspectral stimulated Raman scattering microscopy ensued with multivariate curve resolution analysis to spectrally decompose S. aureus biofilm into its major components (i.e., bacteria and extracellular polymeric substances). Concurrently, vancomycin is conjugated with aryl-alkyne Raman tag (Raman peak at 2218 cm-1) for in vivo tracking of its uptake into biofilm without tissue interference. Results: We find that vancomycin penetration is a non-uniform diffusion process with penetration depths limited by the preferential affinity to the cell clusters. Semi-quantitative analysis shows that the majority of vancomycin binds to the bacteria, achieving intracellular concentrations of up to 4- to 10- fold higher than the administered dosage. The diffusion constant of ~3.16 µm2/min based on the diffusion and antibiotic binding equations is obtained that well accounts for the antibiotic penetration into the biofilm. SRS longitudinal monitoring of antibiotic effect on the growth of biofilms shows that the antibiotics can eradicate the upper layer of the biofilm exposed to sufficient dosages, while the lower layer of the biofilm at a sub-inhibitory dose remains viable, eventually re-growing to significant bio-volume. Conclusion: The Raman-tagged hyperspectral SRS microscopy developed is a powerful imaging tool for dynamic monitoring of inhibitory effects of antibiotics on the growing biofilm in vivo, which would facilitate the formulation of new antibiotics for more effective treatments of bacterial infections in near future.


Assuntos
Antibacterianos/análise , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Microscopia Óptica não Linear/métodos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Vancomicina/análise
7.
J Mol Model ; 25(3): 54, 2019 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-30734871

RESUMO

Antimicrobial peptides (AMPs) are best known for their bactericidal properties; however, due to their unique and flexible structures, they have also been proposed as potential selective sorbents for specific molecules. In the present study, we aimed to design and produce a new peptide-based microextraction fiber for preconcentrating morphine in urine samples. The binding of morphine to the peptide was first evaluated by computational simulation using the Molecular Operating Environment (MOE) 2015.10 software. A similar study was then performed using DS BIOVIA Materials Studio 2017 v17.1.0.48, which confirmed the results of the simulation carried out with MOE. Afterwards, those results were also confirmed by experimental research. In the experimental evaluation, carbon nanotubes (CNTs) were initially carboxylated with H2SO4/HNO3 (3:1) and then functionalized with the peptide. FTIR analysis, Raman measurements, and SEM imaging were used to confirm that CNT functionalization was successful as well as to check the nanostructure of the fiber. To evaluate the functionality of the fiber, it was inserted into a microtube containing a urine sample that included morphine and then sonicated for 5 min at 40 °C. Afterwards, the fiber was washed with methanol 20% (H2O/methanol) and the resulting sample was analyzed by HPLC. This procedure was repeated for different concentrations of morphine in the urine sample. The computational and experimental results showed that a morphine concentration as low as 0.25 ppb in urine could be adsorbed and detected using the peptide fiber. Therefore, given its semi-selective binding affinity for morphine, this peptide-based fiber can be considered a new approach to the detection of small amounts of morphine in biological samples.


Assuntos
Modelos Moleculares , Morfina/urina , Adsorção , Fracionamento Químico/métodos , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Morfina/química , Nanotubos de Carbono/química , Microscopia Óptica não Linear , Sonicação , Espectroscopia de Infravermelho com Transformada de Fourier , Urinálise/métodos
8.
Sci Total Environ ; 654: 1379-1388, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30527887

RESUMO

Machu Picchu Inca sanctuary (Cusco Region, Peru) was constructed on a granitic plateau, better known as Vilcabamba batholith. One of the most important carved granitic rocks from this archaeological site is the Sacred Rock, used by Inca citizens for religious rituals. Due to the location and climatic conditions, different rocks from this archaeological site are affected by biocolonizations. Concretely, the Sacred Rock shows flaking and delamination problems. In this work, a non-destructive multi analytical methodology has been applied to determine the possible role of the biodeteriogens, forming the biological patina on the Sacred Rock, in the previously mentioned conservation problems. Before characterizing the biological patina, a mineralogical characterization of the granitic substrate was conducted using X-ray Diffraction, Raman microscopy (RM) and micro energy dispersive X-ray fluorescence spectrometry. For the identification of the main biodeteriogens in the biofilm, Phase Contrast Microscopy was used. RM also allowed to determine the distribution (imaging) and the penetration (depth profiling) of the biogenic pigments present in the biopatina. Thanks to this study, it was possible to asses that some colonizers are growing on inner areas of the rock, reinforcing their possible assistance in the delamination. Moreover, the in-depth distribution of a wide variety of carotenoids in the patinas allowed to approach the penetration ability of the main biodeteriogens and the diffusion of these biogenic pigments to the inner areas of the rocky substrate.


Assuntos
Biofilmes/crescimento & desenvolvimento , Cianobactérias/fisiologia , Líquens/fisiologia , Microalgas/fisiologia , Arqueologia , Cianobactérias/isolamento & purificação , Líquens/isolamento & purificação , Microalgas/isolamento & purificação , Microscopia Óptica não Linear , Peru , Pigmentos Biológicos/classificação , Dióxido de Silício , Espectrometria por Raios X , Análise Espectral Raman , Difração de Raios X
9.
Transplant Proc ; 50(10): 3128-3134, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30577178

RESUMO

BACKGROUND: Nonlinear optical microscopic (NLOM) imaging technique shows its high resolution imaging features in histocytology. The purpose of this study was to investigate NLOM imaging technique as a useful tool for a donor kidney quality assessment. MATERIALS AND METHODS: Eighty-three pretransplant kidney biopsies from adult donors were analyzed retrospectively. Each specimen was paraffin-embedded and sectioned into 2 consecutive 5-µm thick sections. One section was stained with Masson trichrome, and the other was left unstained for NLOM imaging using second harmonic generation combined with two-photon excited fluorescence (SHG/TPEF). The pretransplant kidney quality was assessed by an experienced pathologist using the Remuzzi scoring system, which characterizes renal tissue morphology into 4 aspects: tubular atrophy, interstitial fibrosis, glomerulosclerosis, and vascular injury. The K coefficient was used to measure the consistency of the Remuzzi scores between conventional Masson trichrome stained images and SHG/TPEF images. RESULTS: NLOM imaging technology can capture high-resolution tissue images from unstained renal tissue, is easy to operate, and shortens time-consuming histological processing procedures. No significant differences (P > .05) were found between the Remuzzi scores of the SHG/TPEF images and the Masson trichrome stained images. The high κ coefficients (0.804-0.895) showed a good consistency between these 2 techniques. CONCLUSION: The NLOM technique is suitable for renal tissue imaging and could potentially be used for routine pretransplant kidney evaluation in clinical settings.


Assuntos
Seleção do Doador , Nefropatias/diagnóstico por imagem , Nefropatias/patologia , Transplante de Rim , Microscopia Óptica não Linear , Adolescente , Adulto , Biópsia , Feminino , Humanos , Nefropatias/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Coloração e Rotulagem , Adulto Jovem
10.
Sci Adv ; 4(11): eaat7715, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30456301

RESUMO

One of the key pathological features of Alzheimer's disease (AD) is the existence of extracellular deposition of amyloid plaques formed with misfolded amyloid-ß (Aß). The conformational change of proteins leads to enriched contents of ß sheets, resulting in remarkable changes of vibrational spectra, especially the spectral shifts of the amide I mode. Here, we applied stimulated Raman scattering (SRS) microscopy to image amyloid plaques in the brain tissue of an AD mouse model. We have demonstrated the capability of SRS microscopy as a rapid, label-free imaging modality to differentiate misfolded from normal proteins based on the blue shift (~10 cm-1) of amide I SRS spectra. Furthermore, SRS imaging of Aß plaques was verified by antibody staining of frozen thin sections and fluorescence imaging of fresh tissues. Our method may provide a new approach for studies of AD pathology, as well as other neurodegenerative diseases associated with protein misfolding.


Assuntos
Doença de Alzheimer/patologia , Modelos Animais de Doenças , Microscopia Óptica não Linear/métodos , Placa Amiloide/patologia , Doença de Alzheimer/diagnóstico por imagem , Precursor de Proteína beta-Amiloide/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Placa Amiloide/diagnóstico por imagem , Presenilinas/genética
11.
J Vis Exp ; (140)2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30371655

RESUMO

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.


Assuntos
Microscopia Óptica não Linear/métodos , Compostos Orgânicos Voláteis/química
12.
J Mater Sci Mater Med ; 29(11): 161, 2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30357534

RESUMO

Segmented polyurethanes were prepared with polycaprolactone diol as soft segment and various amounts of 4,4´-Methylenebis(cyclohexyl isocyanate) and atorvastatin, a statin used for lowering cholesterol, in order to obtain SPU with different content of rigid segments. Polyurethanes with 35% or 50% of rigid segment content were physicochemically characterized and their biocompatibility assessed with L929 fibroblasts. High concentrations of atorvastatin were incorporated by increasing the content of rigid segments as shown by FTIR, Raman, NMR, XPS and EDX. Thermal and mechanical characterization showed that polyurethanes containing atorvastatin and 35% of rigid segments were low modulus (13 MPa) semicrystalline polymers as they exhibited a glass transition temperature (Tg) at -38 °C, melting temperature (Tm) at 46 °C and crystallinity close to 35.9% as determined by DSC. In agreement with this, X-ray diffraction showed reflections at 21.3° and 23.6° for PCL without reflections for atorvastatin suggesting its presence in amorphous form with higher potential bioavailability. Low content of rigid segments led to highly degradable polymer in acidic, alkaline and oxidative media with an acceptable fibroblast cytotoxicity up to 7 days possibly due to low atorvastatin content.


Assuntos
Atorvastatina/química , Materiais Biocompatíveis/química , Cianatos/química , Poliésteres/química , Poliuretanos/química , Animais , Atorvastatina/toxicidade , Materiais Biocompatíveis/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Estrutura Molecular , Microscopia Óptica não Linear , Poliésteres/toxicidade , Poliuretanos/toxicidade , Espectrofotometria Infravermelho , Temperatura Ambiente
13.
J Biomed Opt ; 23(10): 1-9, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30350492

RESUMO

Stimulated Raman scattering microscopy (SRS) was deployed to quantify enamel demineralization in intact teeth. The surfaces of 15 bovine-enamel blocks were divided into four equal-areas, and chemically demineralized for 0, 8, 16, or 24 h, respectively. SRS images (spectral coverage from ∼850 to 1150 cm - 1) were obtained at 10-µm increments up to 90 µm from the surface to the dentin-enamel junction. SRS intensities of phosphate (peak: 959 cm - 1), carbonate (1070 cm - 1), and water (3250 cm - 1) were measured. The phosphate peak height was divided by the carbonate peak height to calculate the SRS-P/C-ratio, which was normalized relative to 90 µm (SRS-P/C-ratio-normalized). The water intensity against depth decay curve was fitted with exponential decay. A decay constant (SRS-water-content) was obtained. Knoop-hardness values were obtained before (SMHS) and after demineralization (SMHD). Surface microhardness-change (SMH-change) [ ( SMHD - SMHS ) / SMHS] was calculated. Depth and integrated mineral loss (ΔZ) were determined by transverse microradiography. Comparisons were made using repeated-measures of analysis of variance. For SRS-P/C-ratio-normalized, at 0-µm (surface), sound (0-h demineralization) was significantly higher than 8-h demineralization and 24-h demineralization; 16-h demineralization was significantly higher than 24-h demineralization. For SRS-water-content, 24-h demineralization was significantly higher than all other demineralization-groups; 8-h demineralization and 16-h demineralization were significantly higher than 0-h demineralization. SRS-water-content presented moderate-to-strong correlation with SMH-change and weak-to-moderate correlation with depth. These results collectively demonstrate the potential of using SRS microscopy for in-situ chemical analysis of dental caries.


Assuntos
Esmalte Dentário/diagnóstico por imagem , Microscopia Óptica não Linear/métodos , Processamento de Sinais Assistido por Computador , Desmineralização do Dente/diagnóstico por imagem , Animais , Carbonatos/química , Bovinos , Esmalte Dentário/química , Desenho de Equipamento , Dureza , Fosfatos/química , Água/química
14.
Sci Rep ; 8(1): 13638, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206377

RESUMO

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease. Although genetic predisposition and epigenetic factors contribute to the development of NAFLD, our understanding of the molecular mechanism involved in the pathogenesis of the disease is still emerging. Here we investigated a possible role of a microRNAs-STAT3 pathway in the induction of hepatic steatosis. Differentiated HepaRG cells treated with the fatty acid sodium oleate (fatty dHepaRG) recapitulated features of liver vesicular steatosis and activated a cell-autonomous inflammatory response, inducing STAT3-Tyrosine-phosphorylation. With a genome-wide approach (Chromatin Immunoprecipitation Sequencing), many phospho-STAT3 binding sites were identified in fatty dHepaRG cells and several STAT3 and/or NAFLD-regulated microRNAs showed increased expression levels, including miR-21. Innovative CARS (Coherent Anti-Stokes Raman Scattering) microscopy revealed that chemical inhibition of STAT3 activity decreased lipid accumulation and deregulated STAT3-responsive microRNAs, including miR-21, in lipid overloaded dHepaRG cells. We were able to show in vivo that reducing phospho-STAT3-miR-21 levels in C57/BL6 mice liver, by long-term treatment with metformin, protected mice from aging-dependent hepatic vesicular steatosis. Our results identified a microRNAs-phosphoSTAT3 pathway involved in the development of hepatic steatosis, which may represent a molecular marker for both diagnosis and therapeutic targeting.


Assuntos
Envelhecimento/metabolismo , Fígado Gorduroso/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metformina/farmacologia , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Envelhecimento/patologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Estudo de Associação Genômica Ampla , Camundongos , Microscopia Óptica não Linear , Fosforilação/efeitos dos fármacos
15.
Anal Chem ; 90(17): 10249-10255, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30070837

RESUMO

We report the development and implementation of an epi-detected spectral-focusing hyperspectral stimulated Raman scattering (SRS) imaging technique for label-free biomolecular subtyping of glioblastomas (GBMs). The hyperspectral SRS imaging technique developed generates SRS image stacks (from 2800 to 3020 cm-1 at 7 cm-1 intervals) within 30 s through controlling the time delay between the chirped pump and Stokes beams. SRS images at representative Raman shifts (e.g., 2845, 2885, and 2935 cm-1) delineate the biochemical variations and morphological differences between proneural and mesenchymal subtypes of GBMs. Multivariate curve resolution (MCR) analysis on hyperspectral SRS images enables the quantification of major biomolecule distributions in mesenchymal and proneural GBMs. Further principal component analysis (PCA) and linear discriminant analysis (LDA) together with leave-one SRS spectrum-out, cross-validation (LOOCV) yields a diagnostic sensitivity of 96.7% (29/30) and specificity of 88.9% (28/36) for differentiation between mesenchymal and proneural subtypes of GBMs. This study shows great potential of applying hyperspectral SRS imaging technique developed for rapid, label-free molecular subtyping of GBMs in neurosurgery.


Assuntos
Neoplasias Encefálicas/classificação , Glioblastoma/classificação , Microscopia Óptica não Linear/métodos , Análise Espectral Raman/métodos , Humanos , Análise Multivariada , Análise de Componente Principal
16.
Int J Pharm ; 549(1-2): 283-292, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-30077760

RESUMO

Three-dimensional printing (3DP) has the potential to cause a paradigm shift in the manufacture of pharmaceuticals, enabling personalised medicines to be produced on-demand. To facilitate integration into healthcare, non-destructive characterisation techniques are required to ensure final product quality. Here, the use of process analytical technologies (PAT), including near infrared spectroscopy (NIR) and Raman confocal microscopy, were evaluated on paracetamol-loaded 3D printed cylindrical tablets composed of an acrylic polymer (Eudragit L100-55). Using a portable NIR spectrometer, a calibration model was developed, which predicted successfully drug concentration across the range of 4-40% w/w. The model demonstrated excellent linearity (R2 = 0.996) and accuracy (RMSEP = 0.63%) and results were confirmed with conventional HPLC analysis. The model maintained high accuracy for tablets of a different geometry (torus shapes), a different formulation type (oral films) and when the polymer was changed from acrylic to cellulosic (hypromellose, HPMC). Raman confocal microscopy showed a homogenous drug distribution, with paracetamol predominantly present in the amorphous form as a solid dispersion. Overall, this article is the first to report the use of a rapid 'point-and-shoot' approach as a non-destructive quality control method, supporting the integration of 3DP for medicine production into clinical practice.


Assuntos
Acetaminofen/química , Analgésicos não Entorpecentes/química , Impressão Tridimensional , Tecnologia Farmacêutica/métodos , Acetaminofen/administração & dosagem , Resinas Acrílicas/química , Administração Oral , Analgésicos não Entorpecentes/administração & dosagem , Composição de Medicamentos , Excipientes/química , Derivados da Hipromelose/química , Microscopia Confocal , Modelos Químicos , Microscopia Óptica não Linear , Espectroscopia de Luz Próxima ao Infravermelho , Comprimidos
17.
Sci Rep ; 8(1): 11804, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30087373

RESUMO

Raman microscopy is a powerful imaging technique for biological materials providing information about chemistry in context with microstructure. A 532 nm laser is often used as excitation source, because high spatial resolution and signal intensity can be achieved. The latter can be controlled by laser power and integration time, whereby high power and long times give good signal to noise ratio. However, most biological materials absorb in the VIS range and fluorescence masking the signal or even sample degradation might be hindering. Here, we show that on lignified plant cell walls even very short integration times and low laser powers induce a change in the ratio of the lignin bands at 1660 and 1600 cm-1. Time series on lignin model compounds revealed this change only in aromatic molecules with two OH-groups, such as coniferyl alcohol. Therefore, we conclude that monolignols are present in the cell wall and responsible for the observed effect. The solvent selectivity of the changes points to a laser induced polymerization process. The results emphasize how crucial careful adjustment of experimental parameters in Raman imaging of biological materials is and show the potential of time series and repeated imaging to get additional insights (e.g. monolignols).


Assuntos
Parede Celular/metabolismo , Lignina/metabolismo , Microscopia Óptica não Linear/métodos , Fenilpropionatos/metabolismo , Picea/metabolismo
18.
PLoS One ; 13(7): e0199695, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29995961

RESUMO

In micropaleontological and paleoclimatological studies based on microfossil morphology and geochemistry, assessing the preservation state of fossils is of the highest importance, as diagenetic alteration invalidates textural features and compromises the correct interpretation of stable isotope and trace elemental analysis. In this paper, we present a novel non-invasive and label-free tomographic approach to reconstruct the three-dimensional architecture of microfossils with submicron resolution based on stimulated Raman scattering (SRS). Furthermore, this technique allows deciphering the three-dimensional (3D) distribution of the minerals within these fossils in a chemically sensitive manner. Our method, therefore, allows to identify microfossils, to chemically map their internal structure and eventually to determine their preservation state. We demonstrate the effectiveness of this method by analyzing several benthic and planktonic foraminifera, obtaining full 3D distributions of carbonate, iron oxide and porosity for each specimen. Subsequently, the preservation state of each microfossil can be assessed using these 3D compositional maps. The technique is highly sensitive, non-destructive, time-efficient and avoids the need for sample pretreatment. Therefore, its predestined application is the final check of the state of microfossils before applying subsequent geochemical analyses.


Assuntos
Foraminíferos/química , Fósseis , Microscopia Óptica não Linear/métodos , Paleontologia/métodos , Carbono/análise , Carbonatos/análise , Compostos Férricos/análise
19.
Anal Chem ; 90(14): 8362-8369, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29894163

RESUMO

The goal of this study was to precisely and unambiguously identify foreign particles in human tissues using a combination of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicrometer spatial resolution. This identification for patient care and research has been traditionally studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction, all with some limitations. We designed a model system of stained and unstained cells that contained birefringent talc particles and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra obtained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely assigned to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micrometer in diameter. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.


Assuntos
Macrófagos/ultraestrutura , Microscopia de Polarização/métodos , Microscopia Óptica não Linear/métodos , Ovário/ultraestrutura , Pleura/ultraestrutura , Talco/análise , Animais , Feminino , Humanos , Camundongos , Modelos Moleculares , Tamanho da Partícula , Pleurodese , Células RAW 264.7
20.
PLoS One ; 13(6): e0199166, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29924825

RESUMO

BACKGROUND: Assessment of severity of liver fibrosis is essential in the management of non-alcoholic fatty liver disease (NAFLD). Second Harmonic Generation (SHG) microscopy is a novel optical tissue imaging system that provides automated quantification of fibrosis based on unique architectural features of collagen. This study aims to develop and validate a SHG-based index for automated staging of liver fibrosis in patients with NAFLD. METHODS: SHG microscopy was performed on archived liver biopsy specimens from 83 patients with NAFLD. A unique algorithm was developed to identify specific SHG parameters that correlated with fibrosis stage. The accuracy of the algorithm was compared against clinical assessment by experienced liver histopathologists using the Brunt fibrosis staging and further validated using the leave-one-out cross-validation method. RESULTS: Mean age of the study cohort was 51.8 ± 11.7 years, with 41% males. A fibrosis index (SHG B-index) was developed comprising 14 unique SHG-based collagen parameters that correlated with severity of NAFLD fibrosis in a continuous fashion. The SHG B-index had excellent correlation with Brunt fibrosis stage (Spearman's correlation 0.820, p<0.001). AUROCs for prediction of Brunt fibrosis stages 1, 2, 3 and 4 were 0.853, 0.967, 0.985 and 0.941 respectively. In the cross-validation analysis, the SHG B-index demonstrated high specificity for diagnosis of all grades of fibrosis. A SHG B-index score of >1.76 had an overall diagnostic accuracy of 98.5% for prediction of presence of bridging fibrosis (Brunt stage ≥3) with sensitivity of 87.5%, specificity 98.0%, positive predictive value 96.6% and negative predictive value 92.6%. CONCLUSION: The SHG B-index is a unique SHG-based index that provides accurate automated assessment of fibrosis stage in NAFLD patients.


Assuntos
Cirrose Hepática/patologia , Microscopia de Fluorescência/métodos , Hepatopatia Gordurosa não Alcoólica/complicações , Microscopia Óptica não Linear/métodos , Adulto , Automação , Biópsia , Colágeno/ultraestrutura , Feminino , Humanos , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença
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