RESUMO
We report a means by which atomic and molecular secondary ions, including cholesterol and fatty acids, can be sputtered through single-layer graphene to enable secondary ion mass spectrometry (SIMS) imaging of untreated wet cell membranes in solution at subcellular spatial resolution. We can observe the intrinsic molecular distribution of lipids, such as cholesterol, phosphoethanolamine and various fatty acids, in untreated wet cell membranes without any labeling. We show that graphene-covered cells prepared on a wet substrate with a cell culture medium reservoir are alive and that their cellular membranes do not disintegrate during SIMS imaging in an ultra-high-vacuum environment. Ab initio molecular dynamics calculations and ion dose-dependence studies suggest that sputtering through single-layer graphene occurs through a transient hole generated in the graphene layer. Cholesterol imaging shows that methyl-ß-cyclodextrin preferentially extracts cholesterol molecules from the cholesterol-enriched regions in cell membranes.
Assuntos
Membrana Celular/metabolismo , Colesterol/análise , Etanolaminas/análise , Ácidos Graxos/análise , Espectrometria de Massa de Íon Secundário/métodos , Diagnóstico por Imagem , Grafite/química , Simulação de Dinâmica Molecular , Análise de Célula Única/métodos , beta-Ciclodextrinas/químicaRESUMO
Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.
Assuntos
Células Ciliadas Auditivas Externas/fisiologia , Ruído , Emissões Otoacústicas Espontâneas/fisiologia , Som , Estimulação Acústica , Animais , Células Ciliadas Auditivas Externas/citologia , Perda Auditiva , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Modelos Animais , Emissões Otoacústicas Espontâneas/genética , Estereocílios/fisiologia , Membrana TectorialRESUMO
Glucose-rich diets shorten the life spans of various organisms. However, the metabolic processes involved in this phenomenon remain unknown. Here, we show that sterol regulatory element-binding protein (SREBP) and mediator-15 (MDT-15) prevent the life-shortening effects of a glucose-rich diet by regulating fat-converting processes in Caenorhabditis elegans. Up-regulation of the SREBP/MDT-15 transcription factor complex was necessary and sufficient for alleviating the life-shortening effect of a glucose-rich diet. Glucose feeding induced key enzymes that convert saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), which are regulated by SREBP and MDT-15. Furthermore, SREBP/MDT-15 reduced the levels of SFAs and moderated glucose toxicity on life span. Our study may help to develop strategies against elevated blood glucose and free fatty acids, which cause glucolipotoxicity in diabetic patients.
Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Fatores de Transcrição/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/genética , Dieta , Sacarose Alimentar/farmacologia , Indução Enzimática/efeitos dos fármacos , Ácidos Graxos Dessaturases/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Glucose/metabolismo , Glucose/farmacologia , Glucose/toxicidade , Interferência de RNA , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Fatores de Transcrição/genéticaRESUMO
We have developed a methodology that analyzes the dimensions and conformal doping profiles in fin field effect transistors (FinFET) using time-of-flight medium energy ion scattering (TOF-MEIS). The structure of a 3D FinFET and As dopant profiles were determined by comprehensive simulations of TOF-MEIS measurements made in three different scattering geometries. The width and height of a FinFET and the As doping profiles in the top, side, and bottom of fin were analyzed simultaneously. The results showed the dimension and conformal doping profile of nanostructures with complex shape can be determined by TOF-MEIS nondestructively, quantitatively, and with subnm depth resolution without any sputtering and matrix effects.
RESUMO
We observed the molecular distribution changes that occurred during the regeneration of fresh zebrafish caudal fins using the recently developed ambient high-resolution mass spectrometry (MS) imaging technique of atmospheric pressure-nanoparticle and plasma-assisted laser desorption ionization (AP-nanoPALDI). AP-nanoPALDI analyses of fresh zebrafish caudal fins revealed that the small molecules, including neurotransmitters, amino acids, lipids, and metabolites of the regenerated area, were more evenly distributed throughout the bony rays and inter-ray mesenchymal tissues compared to the original area in the early stage. Zebrafish caudal fins of less than 200 µm thickness can be very useful for tissue regeneration studies using ambient MS imaging by providing sufficient biomolecular information at the molecular level for wound-healing studies. AP-nanoPALDI imaging was compared with a complementary MS imaging tool, surface sensitive time-of-flight secondary ion MS (ToF-SIMS).
Assuntos
Nadadeiras de Animais/fisiologia , Regeneração/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massa de Íon Secundário/métodos , Peixe-Zebra/fisiologia , Nadadeiras de Animais/metabolismo , Animais , Ouro/química , Nanopartículas Metálicas/química , Peixe-Zebra/metabolismoRESUMO
RATIONALE: Lipids are a key component of atherogenesis. However, their physiological role on the progression of atherosclerosis including plaque vulnerability has not been clearly understood, because of the lack of appropriate tools for chemical assessment. OBJECTIVE: We aimed to develop a label-free chemical imaging platform based on multiplex coherent anti-Stokes Raman scattering (CARS) for the correlative study of the morphology and chemical profile of atherosclerotic lipids. METHODS AND RESULTS: Whole aortas from atherosclerotic apolipoprotein E knock-out mice were en face examined by multiplex CARS imaging and 4 distinctive morphologies of the lipids (intra/extracellular lipid droplets and needle-/plate-shaped lipid crystals) were classified. The chemical profiles of atherosclerotic lipids depending on morphologies were firstly identified from intact atheromatous tissue by multiplex CARS. We demonstrated that needle-/plate-shaped lipid crystals in advanced plaques had undergone a phase shift to the solid state with increased protein contents, implying that lipid modification had occurred beforehand. The validity of lipid-selective multiplex CARS imaging was supported by comparative results from oil red O staining and whole-mount immunohistochemistry. By spatial CARS analysis of atherosclerosis progression, we found greater accumulation of lipid crystals in both the lesser curvature of the aortic arch and the innominate artery. Furthermore, multiplex CARS measurement successfully demonstrated the effect of a drug, statin, on atherosclerotic lipids by showing the change of their chemical profiles. CONCLUSIONS: Multiplex CARS imaging directly provides intact morphologies of atherosclerotic lipids with correlative chemical information, thereby suggesting its potential applications in the investigation of lipid-associated disorders and the preclinical drug screening.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Metabolismo dos Lipídeos , Lipídeos/análise , Microespectrofotometria , Análise Espectral Raman/métodos , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/tratamento farmacológico , Compostos Azo , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/patologia , Corantes , Cristalização , Modelos Animais de Doenças , Progressão da Doença , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Masculino , Camundongos , Camundongos Knockout , Estrutura Molecular , Reprodutibilidade dos Testes , Sinvastatina/farmacologia , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
Silicon nanoclusters have become significant research interest due to their potential application to optoelectronic devices in visible range. We investigate the electronic and optical properties of hydrogenated and nitrogen-passivated silicon nanoclusters using density functional theory calculations. The energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of nanoclusters have varying sizes. They are systematically studied using the conventional local density approximation, the generalized gradient approximation, and the time-dependent density functional theory calculations with hybrid functional. The HOMO-LUMO gap is found to decrease monotonically as the size of nanocluster increases. Introducing one and two nitrogen passivants to a Si29H36 nanocluster, we find that the HOMO-LUMO gap decreases as the number of nitrogen passivants increases. It suggests that multi-nitrogen passivants may enable light emission in visible range from smaller clusters.
RESUMO
New methods to analyze cells and tissues in ambient condition without any harsh chemical fixation or physical freezing and drying are summarized in this report. The first approach, an atmospheric pressure mass spectrometry imaging method, is based on laser ablation in atmospheric pressure assisted by atmospheric plasma and nanomaterials such as nanoparticles and graphene to enhance laser ablation. The second one is based on secondary ion mass spectrometry (SIMS) imaging of live cells in solution capped with single-layer graphene to preserve intact and hydrated biological samples even under ultrahigh vacuum for SIMS bio-imaging in solution.
Assuntos
Espectrometria de Massa de Íon Secundário , Pressão Atmosférica , Grafite , Terapia a Laser , Imagem Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.
Assuntos
Junções Célula-Matriz/metabolismo , Células Endoteliais/citologia , Imagem Molecular/métodos , Ressonância de Plasmônio de Superfície/métodos , Adesão Celular , Comunicação Celular , Divisão Celular , Células Endoteliais/metabolismo , Humanos , Microscopia Confocal , Mitose , Reprodutibilidade dos Testes , Coloração e Rotulagem , Estresse Mecânico , Fatores de TempoRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been a useful tool to profile secondary ions from the near surface region of specimens with its high molecular specificity and submicrometer spatial resolution. However, the TOF-SIMS analysis of even a moderately large size of samples has been hampered due to the lack of tools for automatically analyzing the huge amount of TOF-SIMS data. Here, we present a computational platform to automatically identify and align peaks, find discriminatory ions, build a classifier, and construct networks describing differential metabolic pathways. To demonstrate the utility of the platform, we analyzed 43 data sets generated from seven gastric cancer and eight normal tissues using TOF-SIMS. A total of 87 138 ions were detected from the 43 data sets by TOF-SIMS. We selected and then aligned 1286 ions. Among them, we found the 66 ions discriminating gastric cancer tissues from normal ones. Using these 66 ions, we then built a partial least square-discriminant analysis (PLS-DA) model resulting in a misclassification error rate of 0.024. Finally, network analysis of the 66 ions showed disregulation of amino acid metabolism in the gastric cancer tissues. The results show that the proposed framework was effective in analyzing TOF-SIMS data from a moderately large size of samples, resulting in discrimination of gastric cancer tissues from normal tissues and identification of biomarker candidates associated with the amino acid metabolism.
Assuntos
Íons/química , Espectrometria de Massa de Íon Secundário , Automação , Análise Discriminante , Humanos , Internet , Análise dos Mínimos Quadrados , Neoplasias/metabolismoRESUMO
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging analysis was performed on murine macrophage cells treated with various concentrations of iron oxide (Fe3O4) nanoparticles, which are used as MRI contrast agents. First, murine macrophage cells were seeded on a slide glass for 24 hrs and treated with varying concentrations of Fe3O4 nanoparticles for 24 hrs. To expose a cross section of each cell and obtain a distribution of the nanoparticles inside the cells, the cells were sputtered using Bi ions after which the cross section of each cell was scanned and imaged using the focused cluster ion beam with a spatial resolution of 300 nm. Fe3O4 nanoparticles were found mainly in the cytoplasm region of the cells, not in the nucleus region of cells, suggesting that the uptake of the Fe3O4 nanoparticles were into the cytoplasm of cell, not into the nucleus of cell. Based on these observations, our protocol using mass imaging analysis would be a useful addition to the study of in vitro nanoparticle cytotoxicity.
Assuntos
Compostos Férricos/análise , Macrófagos/química , Nanopartículas Metálicas/análise , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Técnicas Citológicas/métodos , Citoplasma/metabolismo , Compostos Férricos/metabolismo , Compostos Férricos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Camundongos , Espectrometria de Massa de Íon Secundário/métodos , Testes de ToxicidadeRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an imaging-based analytical technique that can characterize the surfaces of biomaterials. We used TOF-SIMS to identify important metabolites and oncogenic KRAS mutation expressed in human colorectal cancer (CRC). We obtained 540 TOF-SIMS spectra from 180 tissue samples by scanning cryo-sections and selected discriminatory molecules using the support vector machine (SVM) algorithm. Each TOF-SIMS spectrum contained nearly 860,000 ion profiles and hundreds of spectra were analyzed; therefore, reducing the dimensionality of the original data was necessary. We performed principal component analysis after preprocessing the spectral data, and the principal components (20) of each spectrum were used as the inputs of the SVM algorithm using the R package. The performance of the algorithm was evaluated using the receiver operating characteristic (ROC) area under the curve (AUC) (0.9297). Spectral peaks (m/z) corresponding to discriminatory molecules used to classify normal and tumor samples were selected according to p-value and were assigned to arginine, α-tocopherol, and fragments of glycerophosphocholine. Pathway analysis using these discriminatory molecules showed that they were involved in gastrointestinal disease and organismal abnormalities. In addition, spectra were classified according to the expression of KRAS somatic mutation, with 0.9921 AUC. Taken together, TOF-SIMS efficiently and simultaneously screened metabolite biomarkers and performed KRAS genotyping. In addition, a machine learning algorithm was provided as a diagnostic tool applied to spectral data acquired from clinical samples prepared as frozen tissue slides, which are commonly used in a variety of biomedical tests.
RESUMO
The ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer's disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-ß (Aß) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aß production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aß secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aß production through an oversupply of cholesterol.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Apolipoproteína E4/genética , Astrócitos/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteína E4/metabolismo , Biomarcadores , Comunicação Celular , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Espaço Extracelular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/efeitos dos fármacosRESUMO
Odor adaptation allows the olfactory system to regulate sensitivity to different stimulus intensities, which is essential for preventing saturation of the cell-transducing machinery and maintaining high sensitivity to persistent and repetitive odor stimuli. Although many studies have investigated the structure and mechanisms of the mammalian olfactory system that responds to chemical sensation, few studies have considered differences in neuronal activation that depend on the manner in which the olfactory system is exposed to odorants, or examined activity patterns of olfactory-related regions in the brain under different odor exposure conditions. To address these questions, we designed three different odor exposure conditions that mimicked diverse odor environments and analyzed c-Fos-expressing cells (c-Fos+ cells) in the odor columns of the olfactory bulb (OB). We then measured differences in the proportions of c-Fos-expressing cell types depending on the odor exposure condition. Surprisingly, under the specific odor condition in which the olfactory system was repeatedly exposed to the odorant for 1 min at 5-min intervals, one of the lateral odor columns and the ipsilateral hemisphere of the olfactory tubercle had more c-Fos+ cells than the other three odor columns and the contralateral hemisphere of the olfactory tubercle. However, this interhemispheric asymmetry of c-Fos expression was not observed in the anterior piriform cortex. To confirm whether the anterior olfactory nucleus pars externa (AONpE), which connects the left and right OB, contributes to this asymmetry, AONpE-lesioned mice were analyzed under the specific odor exposure condition. Asymmetric c-Fos expression was not observed in the OB or the olfactory tubercle. These data indicate that the c-Fos expression patterns of the olfactory-related regions in the brain are influenced by the odor exposure condition and that asymmetric c-Fos expression in these regions was observed under a specific odor exposure condition due to synaptic linkage via the AONpE.
Assuntos
Tubérculo Olfatório/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Olfato/genética , Animais , Encéfalo/metabolismo , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Odorantes , Bulbo Olfatório/metabolismo , Córtex Olfatório/metabolismo , Condutos Olfatórios/citologia , Condutos Olfatórios/metabolismo , Percepção Olfatória/genética , Percepção Olfatória/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Olfato/fisiologiaRESUMO
In spite of recent developments in mass spectrometry imaging techniques, high-resolution multiplex protein bioimaging techniques are required to unveil the complex inter- and intracellular biomolecular interactions for accurate understanding of life phenomena and disease mechanisms. Herein, we report multiplex protein imaging with secondary ion mass spectrometry (SIMS) using metal oxide nanoparticle (MONP)-conjugated antibodies with <300 nm spatial resolution in the low ion dose without ion beam damage because of the high secondary ion yields of the MONPs, which can provide simultaneous imaging of several proteins, especially from cell membranes. We applied our new imaging technique for the study of hippocampal tissue samples from control and Alzheimer's disease (AD) model mice; the proximity of protein clusters in the hippocampus CA1 region showed intriguing dependence on aging and AD progress, suggesting that protein cluster proximity may be helpful for understanding pathological pathways in the microscopic cellular level.
Assuntos
Anticorpos/imunologia , Nanopartículas Metálicas/química , Proteínas/imunologia , Espectrometria de Massa de Íon Secundário/métodos , Doença de Alzheimer/diagnóstico por imagem , Animais , Anticorpos/química , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Região CA1 Hipocampal/diagnóstico por imagem , Região CA1 Hipocampal/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxidos , Tamanho da Partícula , Proteínas/metabolismoRESUMO
We present a three-color multiplex coherent anti-Stokes Raman scattering (CARS) setup that facilitates a prompt recording of broadband CARS spectra along with a fast CARS imaging. With separate narrowband Stokes and probe beams being introduced in the near IR, we are able to incorporate a stable, wideband Ti:sapphire femtosecond laser as a pump beam that covers the full range of Raman shift for CHn stretching vibrational modes. Experimentally, high-resolution multiplex CARS signals are allowed to investigate molecular vibrations over the range of 2650 cm-1 - 3050 cm-1, which are spectrally integrated to construct lipid-sensitive images. It is demonstrated that the proposed implementation promises a particular benefit on CARS imaging of lipid-rich tissue structures by providing detailed information on CHn Raman-active vibrations at points of interest on the CARS images that can be obtained at high frame rates.
Assuntos
Carbono/química , Colorimetria/instrumentação , Hidrogênio/química , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Análise Espectral Raman/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , VibraçãoRESUMO
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool to obtain both chemical information and spatial distribution of specific molecules of interest on a specimen surface. However, since the focused ion beam requires ultrahigh vacuum conditions for desorption and ionization of analytes, proper specimen preparation, such as drying, freeze-drying, and frozen dehydration, is required for ToF-SIMS analysis. In particular, biological specimens with high moisture content generally have a problem of specimen deformation during the normal drying process for a vacuum environment. In this study, the authors propose a cellular specimen preparation method to improve the ion imaging of cells by reducing the deformation of specimens in ToF-SIMS analysis. When the cells on the slide substrate are completely covered with single-layer graphene, the ToF-SIMS imaging is improved by reduced cell deformation due to slow drying. In addition, the graphene encapsulation also induces a reduction in the yield of secondary ions, thereby suppressing the background ion spectra generated by the unwanted organic residues on the substrate, resulting in the improvement of ToF-SIMS imaging. The authors also found that adding plasma treatment to this sample preparation can further improve ion imaging of cells. After cell dehydration is completed, the covered graphene layer can be peeled off by air-plasma treatment and the unwanted organic residues on the substrate can be removed due to plasma cleaning, thereby much improving ion imaging of cells.
Assuntos
Grafite/química , Neurônios , Pele , Animais , Dessecação , Neurônios/química , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Pele/química , Pele/diagnóstico por imagem , Espectrometria de Massa de Íon SecundárioRESUMO
We report on sample preparation methods based on plasma treatment for an improvement of multiple molecular ion images of cellular membranes in the ToF-SIMS method. The air-plasma treatment of fixed cellular samples efficiently removed the organic residues of any solutions used during sample preparation and improved the quality of ToF-SIMS images due to the resulting clean surface. We also studied cell preparation methods that combine single-layer graphene covering with air-plasma treatment to achieve a synergistic effect that eliminates background spectra by organic impurities while minimizing morphological cell deformation in a vacuum environmental analysis. When the cellular sample on the glass substrate is completely covered with the single-layer graphene, the cells trapped between the graphene and the substrate can effectively reduce morphological deformation by slow-dehydration. After slow-dehydration of cells is completed inside the graphene-cover, the covered graphene layer can be peeled off by air-plasma treatment, and the unwanted organic residues on the surface of cells and substrate can also be removed by plasma cleaning, thereby much improving ion imaging of cells with the ToF-SIMS method. It is confirmed that the cell samples in which the graphene-cover was removed by air-plasma treatment maintained their morphology well in comparison with the rapid air-dried cells in atomic force microscopy (AFM) and ToF-SIMS images.
RESUMO
When a tissue slice pretreated with gold nanoparticles is irradiated with a focused 532-nm continuous wave laser, desorption is observed to be uniform and stable, and its shape is sharp and steep. However, since the desorbed molecules are still electrically neutral particles in atmospheric pressure (AP), additional procedure of ionizing them is necessary for AP mass spectrometry (AP-MS) analysis. Therefore, the authors have combined an electrospray device with a simple chamber connected to the airflow-assisted particle transport equipment mounted at the mass spectrometer inlet. Subsequent ionization processes using an electrospray device enable the detection of several types of diacylglycerol molecules above 500 Da, which cannot be detected with the use of AP plasma jets. The authors also developed a remote AP-MS using a long and flexible sampling probe and a fiber laser with a slight modification of the proposed AP desorption and ionization method.
Assuntos
Pressão Atmosférica , Diglicerídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The atmospheric pressure mass spectrometric (AP-MS) imaging technology combined with an inverted optical microscopic system is a powerful tool for determining the presence and spatial distributions of specific biomolecules of interest in live tissues. Efficient desorption and ionization are essential to acquire mass spectrometric (MS) information in an ambient environment. In this study, we demonstrate a new and efficient desorption process using a graphene-coated glass substrate and a continuous wave (CW) laser for high-resolution AP-MS imaging of a live hippocampal tissue. We found that desorption of biomolecules in a live tissue slice was possible with the aid of a graphene-coated glass substrate and indirect application of a 532 nm CW laser on the graphene substrate. Interestingly, the desorption efficiency of a live tissue on the graphene-coated substrate was strongly dependent on the number of graphene layers. Single-layer graphene was found to be the most sensitive substrate for efficient desorption and reproducible high-resolution hippocampal tissue imaging applications. The subsequent ionization process using nonthermal plasma generated sufficient amounts of molecular ions to obtain high-resolution two-dimensional MS images of the cornu ammonis and the dentate gyrus regions of the hippocampus. Therefore, graphene-coated substrates could be a promising platform to induce an efficient desorption process essential for highly reproducible ambient MS imaging.