RESUMO
The temporo-spatial organization of different cells in the tumor microenvironment (TME) is the key to understanding their complex communication networks and the immune landscape that exists within compromised tissues. Multi-omics profiling of single-interacting cells in the native TME is critical for providing further information regarding the reprograming mechanisms leading to immunosuppression and tumor progression. This requires new technologies for biomolecular profiling of phenotypically heterogeneous cells on the same tissue sample. Here, we developed a new methodology for comprehensive lipidomic and metabolomic profiling of individual cells on frozen-hydrated tissue sections using water gas cluster ion beam secondary ion mass spectrometry ((H2O)n-GCIB-SIMS) (at 1.6 µm beam spot size), followed by profiling cell-type specific lanthanide antibodies on the same tissue section using C60-SIMS (at 1.1 µm beam spot size). We revealed distinct variations of distribution and intensities of >150 key ions (e.g., lipids and important metabolites) in different types of the TME individual cells, such as actively proliferating tumor cells as well as infiltrating immune cells. The demonstrated feasibility of SIMS imaging to integrate the multi-omics profiling in the same tissue section at the single-cell level will lead to new insights into the role of lipid reprogramming and metabolic response in normal regulation or pathogenic discoordination of cell-cell interactions in a variety of tissue microenvironments.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Íons , Lipídeos , Espectrometria de Massa de Íon Secundário , Microambiente TumoralRESUMO
Integration of multiomics at the single-cell level allows the unambiguous dissecting of phenotypic heterogeneity at different states such as health, disease, and biomedical response. Imaging mass spectrometry holds the promise of being able to measure multiple types of biomolecules in parallel in the same cell. We have explored the possibility of using water gas cluster ion beam secondary ion mass spectrometry [(H2O)n-GCIB-SIMS] as an analytical tool for multiomics assay. (H2O)n-GCIB has been hailed as an ideal ionization source for biological sampling owing to the enhanced chemical sensitivity and reduced matrix effect. Taking advantage of 1 µm spatial resolution by using a high-energy beam system, we have clearly shown the enhancement of multiple intact biomolecules up to a few hundredfold in single cells. Coupled with the cryogenic sample preparation/measurement, the lipids and metabolites were imaged simultaneously within the cellular region, uncovering the pristine chemistry for integrated omics in the same sample. We have demonstrated that double-charged myelin protein fragments and single-charged multiple lipids and metabolites can be localized in the same cells/tissue with a single acquisition. Our exploration has also been extended to the capability of (H2O)n-GCIB in the generation of multiple charged peptides on protein standards. Frozen hydration combined with (H2O)n-GCIB provides the possibility of universal enhancement for the ionization of multiple bio-molecules, including peptides/proteins which has allowed "omics" to become feasible in the same sample using SIMS.
Assuntos
Espectrometria de Massa de Íon Secundário , Água , Lipídeos , Fenômenos Físicos , ProteínasRESUMO
Peroxidized phosphatidylethanolamine (PEox) species have been identified by liquid chromatography mass spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) imaging with a 70â keV (H2 O)n+ (n>28 000) cluster ion beam, we were able to map PEox with 1.2â µm spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20â pmol µmol-1 lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.
Assuntos
Ferroptose/fisiologia , Peroxidação de Lipídeos/fisiologia , Fosfatidiletanolaminas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Carbolinas/farmacologia , Linhagem Celular , Ferroptose/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-Dawley , Espectrometria de Massa de Íon Secundário/métodosRESUMO
Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.
Assuntos
Metabolômica/métodos , Imagem Óptica/métodos , Purinas/biossíntese , Espectrometria de Massa de Íon Secundário/métodos , Células HeLa , Humanos , Mitocôndrias/metabolismo , Complexos Multienzimáticos/metabolismo , Análise de Célula ÚnicaRESUMO
A gas cluster ion beam (GCIB) source, consisting of CO2 clusters and operating with kinetic energies of up to 60 keV, has been developed for the high resolution and high sensitivity imaging of intact biomolecules. The CO2 molecule is an excellent molecule to employ in a GCIB source due to its relative stability and improved focusing capabilities, especially when compared to the conventionally employed Ar cluster source. Here we report on experiments aimed to examine the behavior of CO2 clusters as they impact a surface under a variety of conditions. Clusters of (CO2)n+ (n = 2000~10,000) with varying sizes and kinetic energies were employed to interrogate both an organic and inorganic surface. The results show that C-O bond dissociation did not occur when the energy per molecule is less than 5 eV/n, but that oxygen adducts were seen in increasing intensity as the energy is above 5 eV/n, particularly, drastic enhancement up to 100 times of oxygen adducts was observed on Au surface. For Irganox 1010, an organic surface, oxygen containing adducts were observed with moderate signal enhancement. Molecular dynamics computer simulations were employed to test the hypothesis that the C-O bond is broken at high values of eV/n. These calculations show that C-O bond dissociation occurs at eV/n values less than the C-O bond energy (8.3 eV) by interaction with surface topological features. In general, the experiments suggest that the projectiles containing oxygen can enhance the ionization efficiency of surface molecules via chemically induced processes, and that CO2 can be an effective cluster ion source for SIMS experiments. Graphical Abstract.
RESUMO
Gas cluster ion beams (GCIBs) provide new opportunities for bioimaging and molecular depth profiling with secondary ion mass spectrometry (SIMS). These beams, consisting of clusters containing thousands of particles, initiate desorption of target molecules with high yield and minimal fragmentation. This review emphasizes the unique opportunities for implementing these sources, especially for bioimaging applications. Theoretical aspects of the cluster ion/solid interaction are developed to maximize conditions for successful mass spectrometry. In addition, the history of how GCIBs have become practical laboratory tools is reviewed. Special emphasis is placed on the versatility of these sources, as size, kinetic energy, and chemical composition can be varied easily to maximize lateral resolution, hopefully to less than 1 micron, and to maximize ionization efficiency. Recent examples of bioimaging applications are also presented.
Assuntos
Gases/química , Fosfolipídeos/análise , Espectrometria de Massa de Íon Secundário/métodos , Animais , Humanos , Íons/química , Simulação de Dinâmica MolecularRESUMO
Obtaining a comprehensive grasp of the behavior and interaction of pharmaceutical compounds within single cells provides some of the fundamental details necessary for more effective drug development. In particular, the changes ensuing in the carrier, drug, and host environment in targeted drug therapy applications must be explored in greater detail, as these are still not well understood. Here, nilotinib-functionalized gold nanoparticles are examined within single mammalian cells with use of imaging cluster secondary ion mass spectrometry in a model study designed to enhance our understanding of what occurs to these particles once that have been internalized. Nilotinib, several types of gold nanoparticles, and the functionalized combination of the two were surveyed and successfully imaged within single cells to determine uptake and performance. Both nilotinib and the gold particle are able to be distinguished and visualized in the functionalized nanoparticle assembly within the cell. These compounds, while both internalized, do not appear to be present in the same pixels of the chemical image, indicating possible cleavage of nilotinib from the particle after cell uptake. The method provided in this work is a direct measurement of uptake and subcellular distribution of an active drug and its carrier within a framework. The results obtained from this study have the potential to be applied to future studies to provide more effective and specific cellular delivery of a relevant pharmaceutical compound.
Assuntos
Antineoplásicos/farmacocinética , Portadores de Fármacos/análise , Ouro/análise , Nanopartículas Metálicas/análise , Pirimidinas/farmacocinética , Espectrometria de Massa de Íon Secundário/métodos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Camundongos , Tamanho da Partícula , Pirimidinas/administração & dosagem , Pirimidinas/química , Células RAW 264.7RESUMO
Gas cluster ion beam-secondary ion mass spectrometry (GCIB-SIMS) has shown the full potential of mapping intact lipids in biological systems with better than 10 µm lateral resolution. This study investigated further the capability of GCIB-SIMS in imaging high-mass signals from intact cardiolipin (CL) and gangliosides in normal brain and the effect of a controlled cortical impact model (CCI) of traumatic brain injury (TBI) on their distribution. A combination of enzymatic and chemical treatments was employed to suppress the signals from the most abundant phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and enhance the signals from the low-abundance CLs and gangliosides to allow their GCIB-SIMS detection at 8 and 16 µm spatial resolution. Brain CLs have not been observed previously using other contemporary imaging mass spectrometry techniques at better than 50 µm spatial resolution. High-resolution images of naive and injured brain tissue facilitated the comparison of CL species across three multicell layers in the CA1, CA3, and DG regions of the hippocampus. GCIB-SIMS also reliably mapped losses of oxidizable polyunsaturated CL species (but not the oxidation-resistant saturated and monounsaturated gangliosides) to regions including the CA1 and CA3 of the hippocampus after CCI. This work extends the detection range for SIMS measurements of intact lipids to above m/z 2000, bridging the mass range gap compared with MALDI. Further advances in high-resolution SIMS of CLs, with the potential for single cell or supra-cellular imaging, will be essential for the understanding of CL's functional and structural organization in normal and injured brain.
Assuntos
Encéfalo/metabolismo , Cardiolipinas/metabolismo , Lasers de Gás , Espectrometria de Massa de Íon Secundário/métodos , Animais , Encéfalo/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/veterinária , Imidas/química , Masculino , Propilaminas/química , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fosfolipases Tipo C/metabolismoRESUMO
In the context of a secondary ion mass spectrometry (SIMS) experiment, dynamic reactive ionization (DRI) involves introducing a reactive dopant, HCl, into an Ar gas cluster primary ion beam along with a source of water to enable dissociation of HCl to free protons. This concerted effect, precisely occurring at the impact site of the cluster beam, enhances the protonation of molecular species. Here, the authors apply this methodology to study the hippocampus and cerebellum region of a frozen-hydrated mouse brain section. To determine the degree of enhancement associated with DRI conditions, sequential tissue slices were arranged in a mirrored configuration so that comparable regions of the tissue could be explored. The results show that the protonated lipid species are increased by â¼10-fold, but that the normally prevalent salt adducts are virtually unaffected. This observation is discussed as a novel approach to minimizing SIMS matrix effects in complex materials. Moreover, the chemical images of protonated lipid ions exhibit clearer features in the cerebellum region as compared to images acquired with the pure Ar cluster beam.
Assuntos
Cerebelo/anatomia & histologia , Cerebelo/química , Hipocampo/anatomia & histologia , Hipocampo/química , Imagem Óptica/métodos , Manejo de Espécimes/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Lipídeos/análise , Camundongos , Sais/análiseRESUMO
In order to utilize complementary imaging techniques to supply higher resolution data for fusion with secondary ion mass spectrometry (SIMS) chemical images, there are a number of aspects that, if not given proper consideration, could produce results which are easy to misinterpret. One of the most critical aspects is that the two input images must be of the same exact analysis area. With the desire to explore new higher resolution data sources that exists outside of the mass spectrometer, this requirement becomes even more important. To ensure that two input images are of the same region, an implementation of the insight segmentation and registration toolkit (ITK) was developed to act as a preprocessing step before performing image fusion. This implementation of ITK allows for several degrees of movement between two input images to be accounted for, including translation, rotation, and scale transforms. First, the implementation was confirmed to accurately register two multimodal images by supplying a known transform. Once validated, two model systems, a copper mesh grid and a group of RAW 264.7 cells, were used to demonstrate the use of the ITK implementation to register a SIMS image with a microscopy image for the purpose of performing image fusion.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imagem Multimodal/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Linhagem Celular , Cobre , Macrófagos , CamundongosRESUMO
To achieve successful drug delivery via nanoparticles the interactions between the nanoparticle and the chemistry of the surrounding biological environment is of central importance. A thorough understanding of these interactions is necessary in order to better elucidate information regarding drug pathways and mechanisms of action in treatment protocols. As such, it is important to identify the location of the nanoparticle, the state of its functionalization, as well as any changes in the cellular environment. The use of cluster secondary ion mass spectrometry (SIMS) using C60 (+) primary ions makes simultaneous acquisition of this information possible. Here, SIMS has been successfully used to chemically image gold nanoparticles (AuNPs) within a model, single cell system involving macrophage-like RAW 264.7 cells. The macrophage-like properties of this cell line make it extremely well-suited for cell-uptake studies. Both AuNPs and two pharmaceutical compounds, amiodarone and elacridar, were successfully imaged within a cellular system using cluster SIMS. To verify that SIMS can also be used to detect functionalization and nanoparticles simultaneously, fluorophore-functionalized AuNPs were studied as a model system. The fluorescent characteristics of these functionalized nanoparticles enabled the visual confirmation of the presence and location of the particles within the cell.
Assuntos
Portadores de Fármacos/análise , Fulerenos/análise , Processamento de Imagem Assistida por Computador/métodos , Macrófagos/química , Nanopartículas/análise , Espectrometria de Massa de Íon Secundário/métodos , Acridinas/análise , Amiodarona/análise , Animais , Antiarrítmicos/análise , Ouro/análise , Camundongos , Células RAW 264.7 , Coloração e Rotulagem/métodos , Tetra-Hidroisoquinolinas/análiseRESUMO
An Ar n+ (n = 1-6000) gas cluster ion source has been utilized to map the chemical distribution of lipids in a mouse brain tissue section. We also show that the signal from high mass species can be further enhanced by doping a small amount of CH4 into the Ar cluster to enhance the ionization of several biologically important molecules. Coupled with secondary ion mass spectrometry instrumentation which utilizes a continuous Ar cluster ion projectile, maximum spatial resolution and maximum mass resolution can be achieved at the same time. With this arrangement, it is possible to achieve chemically resolved molecular ion images at the 4-µm resolution level. The focused Ar n+/[Ar x (CH4) y ]+ beams (4-10 µm) have been applied to the study of untreated mouse brain tissue. A high signal level of molecular ions and salt adducts, mainly from various phosphocholine lipids, has been seen and directly used to map the chemical distribution. The signal intensity obtained using the pure Ar cluster source, the CH4-doped cluster source and C60 is also presented.
RESUMO
Although the benefits of decreased sample temperature for the molecular profiling of organic materials with time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been established, the mechanism behind spectral changes observed at low temperature, particularly increased protonated molecular ion (M + H)(+) yields, have not been examined in detail. We have developed a procedure to investigate these effects by monitoring secondary ion yields under sustained primary ion bombardment as the sample temperature is cooled from room temperature down to 80 K. Examination of biomaterials such as an amino acid (arginine), a polypeptide (Gly-Gly-Tyr-Arg), a lipid (1,2 dipalmitoyl-sn-glycero-3 phosphatidylcholine), and a drug molecule (cyclosporine A) each provide evidence of ion yield enhancement at 80 K under either 20 keV C(60)(+) or 20 keV Au(3)(+) bombardment. For example, arginine shows a 2-fold increase in the steady-state intensity for the (M + H)(+) ion at 80 K compared to the steady state at 300 K. It is shown that there is a correlation between the yield enhancement and a reduction in the damage cross section, which for arginine under 20 keV Au(3)(+) bombardment decreases from 5.0 ± 0.4 × 10(-14) cm(2) at 300 K to 2.0 ± 0.3 × 10(-14) cm(2) at 80 K. The role of water as the facilitator for this reduction is explored through the use of H(2)O and D(2)O dosing experiments at 80 K.
Assuntos
Espectrometria de Massa de Íon Secundário/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Arginina/química , Ciclosporina/química , Óxido de Deutério/química , Oligopeptídeos/química , TemperaturaRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60(+)/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.
Assuntos
Colesterol/análise , Espaço Intracelular/química , Espectrometria de Massas em Tandem/métodos , Colesterol/química , Macrófagos/citologia , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSION: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.
Assuntos
Movimento Celular/fisiologia , Colesterol/metabolismo , Células Germinativas/citologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Animais , Apoptose , Sobrevivência Celular , Embrião de Mamíferos/metabolismo , Feminino , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.
Assuntos
Membrana Celular/química , Colesterol/análise , Diagnóstico por Imagem/métodos , Espectrometria de Massa de Íon Secundário/métodos , Técnicas Citológicas , Humanos , Macrófagos , Microscopia de FluorescênciaRESUMO
Emission of benzene molecules by 5-keV cluster bombardment of a range of carbon projectiles from C6H6 to C180 is studied by a coarse-grained molecular dynamics (MD) technique. This approach permits calculations that are not feasible using more complicated potential energy functions, particularly as the interesting physics associated with the ion impact event approaches the mesoscale. These calculations show that the highest ejection yields are associated with clusters that deposit their incident energy 15-20 A below the surface. The highest yield for the projectiles is produced by the C20 and C60 projectiles. The results from the MD simulations are also compared favorably to an analytical model based on fluid dynamics to describe the energy deposition. The analytical model is then utilized to extend the range of the calculations to higher incident energies. The issue of the relative amount of chemical fragmentation and intact molecular desorption is also examined for the benzene crystal. These results show that damage accumulation at high-incident fluence should not be problematic and that it should be possible to perform molecular depth profiling via secondary ion mass spectrometry experiments. In general, the approach presented here illustrates the power of combining a simplified MD method with analytical strategies for describing a length scale that is difficult to achieve with traditional MD calculations.
Assuntos
Benzeno/química , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Análise por Conglomerados , TermodinâmicaRESUMO
Peptide-doped trehalose thin films have been characterized by bombardment with energetic cluster ion beams of C60+ and Aux+ (x = 1, 2, 3). The aim of these studies is to acquire information about the molecular sputtering process of the peptide and trehalose by measurement of secondary ion mass spectra during erosion. This system is important since uniform thin films of approximately 300 nm thickness can be reproducibly prepared on a Si substrate, allowing detailed characterization of the resulting depth profile with different projectiles. The basic form of the molecular ion intensity as a function of ion dose is described by a simple analytical model. The model includes parameters such as the molecular sputtering yield, the damage cross section of the trehalose or the peptide, and the thickness of a surface layer altered by the projectile. The results show that favorable conditions for successful molecular depth profiling are achieved when the total sputtering yield is high and the altered layer thickness is low. Successful molecular depth profiles are achieved with all of the cluster projectiles, although the degree of chemical damage accumulation was slightly lower with C60. With C60 bombardment, the altered layer thickness of about 20 nm and the damage cross section of about 5 nm2 are physically consistent with predictions of molecular dynamics calculations available for similar chemical systems. In general, the model presented should provide guidance in optimizing experimental parameters for maximizing the information content of molecular depth profiling experiments with complex molecular thin film substrates.
Assuntos
Peptídeos/química , Trealose/química , Algoritmos , Radioisótopos de Cobalto , Ouro , Microscopia de Força Atômica , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
A novel, trehalose-glycerol matrix was utilized to generate high-resolution, TOF-SIMS images of macrophages and glial cells. Viable cells incubated in 50 mM trehalose, then lyophilized in a 50 mM trehalose, 10-15% (w/w) glycerol rinse, are preserved and chemically profiled. These experiments demonstrate the utility of the disaccharide matrix as an efficient, cost-effective alternative to cryogenics for SIMS and other ultrahigh-vacuum (UHV) analyses of biological species. Cellular processes on oligodendrocytes and astrocytes, 1-3 mum in width, were well resolved for cells in the trehalose-glycerol matrix. The viscous cell matrixes were fractured and analyzed at room temperature and maintained their three-dimensional integrity under UHV. Images have been generated with a Au primary ion source near the static limit of 10(12) ions/cm2. Though these nucleated cells do not remain viable after desiccation, TOF-SIMS imaging and subsequent rehydration reveals structural and morphological preservation. Eliminating the inherent obstacles associated with cryogenic analysis opens the door to greater utility of SIMS as a bioanalytical tool, such as lipid mapping of single cells in the nervous system.
Assuntos
Células Eucarióticas/ultraestrutura , Macrófagos/ultraestrutura , Espectrometria de Massas/métodos , Neuroglia/ultraestrutura , Trealose/química , Animais , Liofilização , Glicerol/química , Masculino , Camundongos , RatosRESUMO
A buckminsterfullerene ion source is employed to characterize peptide-doped trehalose thin films. The experiments are designed to utilize the unique sputtering properties of cluster ion beams for molecular depth profiling. The results show that trehalose films with high uniformity can be prepared on Si by a spin-coating technique. Bombardment of the film with C60+ results in high quality time-of-flight secondary ion mass spectrometry spectra, even during ion doses of up to 3 x 10(14) ions/cm2. This result is in contrast to atomic bombardment experiments in which the dose of incident ions must be kept below 10(12) ions/cm2 so as to retain mass spectral information. Moreover, since the films are of uniform thickness, it is possible to depth-profile through the film and into the Si substrate. This experimental protocol allows the yield of trehalose molecular equivalents and the degree of interface mixing to be evaluated in detail. When doped with a variety of small peptides up to a molecular weight of m/z 500, we find that the peptide molecular ion intensity remains stable under continuous C60+ bombardment, although some decrease in intensity is observed. The results are interpreted in terms of a model whereby the high trehalose yield and low damage depth of the C60 projectile combine to prevent damage accumulation. In general, the peptide-trehalose system provides a valuable model for evaluating the parameters that lead to effective 3-dimensional characterization of biomaterials.