RESUMO
Characterization of lignocellulosic biomass microstructure with chemical specificity and under physiological conditions could provide invaluable insights to our understanding of plant tissue development, microstructure, origins of recalcitrance, degradation, and solubilization. However, most methods currently available are either destructive, are not compatible with hosting a physiological environment, or introduces exogenous probes, complicating their use for studying changes in microstructure and mechanisms of plant development, recalcitrance, or degradation in situ. To address these challenges, we here present a multi-modal chemically specific imaging technique based on coherent anti-Stokes Raman scattering (CARS) microspectroscopy with simplex maximization and entropy-based spectral unmixing enabling label-free, chemically specific characterization of plant microstructure in liquid. We describe how spatial drift of samples suspended in liquid can introduce artifacts in spectral unmixing procedures for single-frequency CARS and propose a mitigative strategy toward these effects using simultaneously acquired forward-scattered CARS signals and epi-detected autofluorescence. We further apply the technique for chemical and microstructural characterization of untreated and liquid hot water pretreated rapeseed straw by CARS and show how the framework can be extended for 3D imaging with chemical specificity. Finally, we provide examples of the intricate chemical and microstructural details recovered by this hybrid imaging technique, including discerning between primary and secondary cell walls, localization of aqueous components to cell lumina, and the presence of funnel-type pits in samples ofBrassica napus.
Assuntos
Microscopia , Plantas , Biomassa , Biopolímeros , Microscopia/métodos , Análise Espectral Raman/métodosRESUMO
Current techniques for monitoring disease progression and testing drug efficacy in animal models of inflammatory arthritis are either destructive, time-consuming, subjective, or require ionizing radiation. To accommodate this, we have developed a non-invasive and label-free optical system based on Raman spectroscopy for monitoring tissue alterations in rodent models of arthritis at the biomolecular level. To test different sampling geometries, the system was designed to collect both transmission and reflection mode spectra. Mice with collagen antibody-induced arthritis and controls were subject to in vivo Raman spectroscopy at the tibiotarsal joint every 3 days for 14 days. Raman-derived measures of bone content correlated well with micro-computed tomography bone mineral densities. This allowed for time-resolved quantitation of bone densities, which indicated gradual bone erosion in mice with arthritis. Inflammatory pannus formation, bone erosion, and bone marrow inflammation were confirmed by histological analysis. In addition, using library-based spectral decomposition, we quantified the progression of bone and soft tissue components. In general, the tissue components followed significantly different tendencies in mice developing arthritis compared to the control group in line with the histological analysis. In total, this demonstrates Raman spectroscopy as a versatile technique for monitoring alterations to both mineralized and soft tissues simultaneously in rodent models of musculoskeletal disorders. Furthermore, the technique presented herein allows for objective repeated within-animal measurements potentially refining and reducing the use of animals in research while improving the development of novel antiarthritic therapeutics.
Assuntos
Artrite , Análise Espectral Raman , Camundongos , Animais , Microtomografia por Raio-X/métodos , Análise Espectral Raman/métodos , Modelos Animais , Progressão da Doença , Modelos Animais de DoençasRESUMO
Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.
Assuntos
Materiais Biocompatíveis/química , Substitutos Ósseos/química , Estrôncio/química , Regeneração Óssea , Cerâmica/química , Colesterol/química , Meios de Cultivo Condicionados/química , Vidro/química , Humanos , Lipídeos/química , Teste de Materiais , Microdomínios da Membrana , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácido Mevalônico/química , Análise em Microsséries , Miosinas/química , Fosforilação , Proteínas/química , RNA Mensageiro/metabolismo , Análise Espectral Raman , Regulação para CimaRESUMO
We report on the use of high resolution Raman spectroscopy mapping combined with a micro-engineered stem cell platform. This technique obtains quantitative information about the concentration of individual intracellular molecules such as proteins, lipids, and other metabolites, while tightly controlling cell shape and adhesion. This new quantitative analysis will prove highly relevant for in vitro drug screening applications and regenerative medicine.
Assuntos
Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Análise Espectral Raman/métodos , Linhagem Celular , Forma Celular , Humanos , Lipídeos/análise , Proteínas/análiseRESUMO
In vitro Raman spectroscopy used for non-invasive, non-destructive characterization of single cells and tissues has proven to be a powerful tool for understanding the complex biochemical processes within these biological systems. Additionally it enables the comparison of a wide range of in vitro model systems by discriminating them based on their biomolecular differences. However, one persistent challenge in Raman spectroscopy has been the highly complex structure of cell and tissue spectra, which comprise signals from lipids, proteins, carbohydrates and nucleic acids, which may overlap significantly. This leads to difficulty in discerning which molecular components are responsible for the changes seen between experimental groups. To address this problem, we introduce a technique to highlight the significant biochemical changes between sample groups by applying a novel approach using Partial Least Squares - Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores normally used for variable selection as heat maps combined with group difference spectra to highlight significant differences in Raman band shapes and position. To illustrate this method we analyzed single HeLa cells in their live, fixed, fixed and ethanol dehydrated, to the fixed, dehydrated and then rehydrated states respectively. Fixation, ethanol dehydration and rehydration are known to induce molecular changes in the lipids and proteins within each cell.
Assuntos
Análise Espectral Raman/métodos , Análise Discriminante , Células HeLa , Humanos , Análise dos Mínimos Quadrados , Fixação de TecidosRESUMO
Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.
Assuntos
Proteólise/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Alicerces Teciduais/química , Proteína com Valosina/antagonistas & inibidores , Fenômenos Biofísicos , Regeneração Óssea/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Tumoral , Desenvolvimento de Medicamentos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Engenharia Tecidual/métodosRESUMO
OBJECTIVE: Craniofacial bone trauma is a leading reason for surgery at most hospitals. Large pieces of destroyed or resected bone are often replaced with non-resorbable and stock implants, and these are associated with a variety of problems. This paper explores the use of a novel fatty acid/calcium phosphate suspension melt for simple additive manufacturing of ceramic tricalcium phosphate implants. METHODS: A wide variety of non-aqueous liquids were tested to determine the formulation of a storable 3D printable tricalcium phosphate suspension ink, and only fatty acid-based inks were found to work. A heated stearic acid-tricalcium phosphate suspension melt was then 3D printed, carbonized and sintered, yielding implants with controllable macroporosities. Their microstructure, compressive strength and chemical purity were analyzed with electron microscopy, mechanical testing and Raman spectroscopy, respectively. Mesenchymal stem cell culture was used to assess their osteoconductivity as defined by collagen deposition, alkaline phosphatase secretion and de-novo mineralization. RESULTS: After a rapid sintering process, the implants retained their pre-sintering shape with open pores. They possessed clinically relevant mechanical strength and were chemically pure. They supported adhesion of mesenchymal stem cells, and these were able to deposit collagen onto the implants, secrete alkaline phosphatase and further mineralize the ceramic. SIGNIFICANCE: The tricalcium phosphate/fatty acid ink described here and its 3D printing may be sufficiently simple and effective to enable rapid, on-demand and in-hospital fabrication of individualized ceramic implants that allow clinicians to use them for treatment of bone trauma.
Assuntos
Regeneração Óssea , Fosfatos de Cálcio , Osso e Ossos , Cerâmica , Força Compressiva , Porcelana Dentária , Células-Tronco MesenquimaisRESUMO
Imaging by Raman spectroscopy enables unparalleled label-free insights into cell and tissue composition at the molecular level. With established approaches limited to single image analysis, there are currently no general guidelines or consensus on how to quantify biochemical components across multiple Raman images. Here, we describe a broadly applicable methodology for the combination of multiple Raman images into a single image for analysis. This is achieved by removing image specific background interference, unfolding the series of Raman images into a single dataset, and normalisation of each Raman spectrum to render comparable Raman images. Multivariate image analysis is finally applied to derive the contributing 'pure' biochemical spectra for relative quantification. We present our methodology using four independently measured Raman images of control cells and four images of cells treated with strontium ions from substituted bioactive glass. We show that the relative biochemical distribution per area of the cells can be quantified. In addition, using k-means clustering, we are able to discriminate between the two cell types over multiple Raman images. This study shows a streamlined quantitative multi-image analysis tool for improving cell/tissue characterisation and opens new avenues in biomedical Raman spectroscopic imaging.
Assuntos
Processamento de Imagem Assistida por Computador , Células-Tronco Mesenquimais/citologia , Análise Espectral Raman , Células Cultivadas , Análise por Conglomerados , Humanos , Análise MultivariadaRESUMO
The formation of insoluble ß-sheet-rich protein structures known as amyloid fibrils is associated with numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. A detailed understanding of the molecular structure of the fibril surface is of interest as the first contact with the physiological environment in vivo and plays a decisive role in biological activity and associated toxicity. Recent studies reveal that the inherent sensitivity and specificity of tip-enhanced Raman scattering (TERS) renders this technique a compelling method for fibril surface analysis at the single-particle level. Here, the reproducibility of TERS is demonstrated, indicating its relevance for detecting molecular variations. Consequently, individual fibrils are systematically investigated at nanometer spatial resolution. Spectral parameters were obtained by band-fitting, particularly focusing on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest.
Assuntos
Amiloide/química , Interações Hidrofóbicas e Hidrofílicas , Insulina/química , Domínios e Motivos de Interação entre Proteínas , Análise Espectral , Aminoácidos/química , Amiloide/metabolismo , Insulina/metabolismo , Microscopia de Força Atômica , Análise Espectral/métodos , Análise Espectral RamanRESUMO
Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology.
Assuntos
Temperatura Alta , Intestino Delgado/citologia , Intestino Delgado/cirurgia , Ondas de Rádio , Análise Espectral Raman , Animais , Intestino Delgado/efeitos da radiação , Microscopia , SuínosRESUMO
BACKGROUND: To visualise and characterise skin architecture, the tissue usually has to be destroyed and labelled. OBJECTIVES: The use of Fourier transform infrared (FTIR) spectroscopy as a label-free, minimally sample destructive method to define hair follicular structure has been explored and demonstrated in this paper. METHODS: Human scalp skin cryosections were imaged using FTIR microscopy and the data was subsequently analysed with N-FINDR spectral unmixing algorithm. RESULTS: This resulted in an excellent distinction of known hair follicle tissue layers, which could be discerned based on their molecular structure. CONCLUSION: The development of a minimally sample-destructive, label-free spectroscopy based technique that can differentiate layers of cells in the dermal papilla and connective tissue sheath in the mesenchyme of the hair follicle paves the way forward to identifying spectral markers important in wound healing and stem cell therapies.