Background
Knowledge about the molecular-level composition and processes of cells and tissues forms the basis of modern biology and biomedicine. Advances in this area are critical for the development of new drugs and improved diagnostic and therapeutic methods, and also for opening up new potential applications in biotechnology. Ideally, a full characterization of a biological sample should provide information about all substances present and their concentrations, as well as their spatial distribution or localization. Presently there is a lack of analytical methods for precise identification and localisation of chemical substances at high spatial (subcellular) resolution.
Current state-of-the-art techniques for biochemical (molecular) analysis of cells or tissues are based on either a non-local analysis or imaging analysis of pre-selected and labelled substances. In the first approach, elaborately prepared samples are analysed by different techniques, such as gel electrophoresis, mass spectrometry (GC-MS, LC-MS-MS, MALDI-MS), optical spectroscopy, or microarrays. The spatial information is limited to what can be achieved in the sampling process, which may include micro-dissection or micro-dialysis. Obviously, this approach has no imaging capability, i.e., it does not yield any structural information about intact biological systems. The second approach is based on staining techniques or the use of labelled molecules that bind specifically to pre-selected target molecules (or tissue structures). Radioactive labels, fluorescent labels and colloidal labels are detected by radiography, fluorescence microscopy and electron microscopy, respectively. These techniques allow localisation, in the form of images, of selected target molecules with lateral resolutions normally in the range 0.4-10 µm. With confocal microscopes, 3D-information can be obtained with a depth resolution down to 500 nm. Electron microscopy can be used to map the localisation of single colloid-labelled molecules with a lateral resolution down to ~1 nm. The main limitations of this approach are that it relies on pre-selection of the molecules to be detected and that specific markers for many relevant biomolecules are unavailable, making co-localisation of different classes of molecules difficult or even impossible.
The NANOBIOMAPS project aims at significantly improving the possibilities for imaging analysis of biological samples, compared to present state-of-the-art techniques. The overall objective is to develop new techniques for sub-micrometer-resolved mapping of organic and inorganic substances in biological samples, from single cells to organised tissues. The techniques will be based on time-of-flight secondary ion mass spectrometry (TOF-SIMS) and laser secondary neutral mass spectrometry (laser-SNMS), providing parallel identification, localisation and co-localisation of a variety of substances without the need for pre-selected molecular labelling.
The specific goals and anticipated output of the project are:
1. Optimisation of the spatial resolution, obtaining localisation of specific substances in cell and tissue samples at a spatial resolution of < 50 nm.
2. Development of methods for 3D mapping of specific substances in cells and tissue with a depth resolution of 1-10 nm (lateral resolution <50 nm). Different intracellular locations/levels and organelles in the biological samples will be accessed by several methods, including mechanical sectioning and ion etching.
3. Simultaneous and label-free identification, localisation and co-localisation of a broad range of substances, such as membrane lipids, peptides and proteins, drugs, and metabolites. The identification of >100 new unlabeled substances not yet identified by TOF-SIMS and laser-SNMS in biological specimens will be achieved.
Another goal of the project is to contribute to establishing TOF-SIMS and laser-SNMS as useful techniques in the life sciences and making them widely known to a large group of potential users in the biological and biomedical research communities. Furthermore, NANOBIOMAPS will establish Europe as the scientific and technological leader in the development and application of this new technology in the biomedical field.
Imaging mass spectrometry, TOF-SIMS, and laser-SNMS
Mass spectrometry methods with imaging capabilities have the potential to simultaneously provide, in one single measurement, the information obtained by both of the approaches described above. A technique based on matrix assisted laser desorption ionization mass spectrometry MALDI-MS has been developed for imaging the distribution of proteins and other substances in tissue. The potential for high-resolution imaging by MALDI-MS is, however, limited by the necessity to use analyte-specific matrices in the sample preparation and by its lateral resolution of ~10 µm, which is insufficient for imaging at a sub-cellular level.
NANOBIOMAPS will develop imaging mass spectrometry technology based on two related methods; time-of-flight secondary ion mass spectrometry (TOF-SIMS) and laser post-ionisation secondary neutral mass spectrometry (laser SNMS). With both techniques, the sample is bombarded with a focused, energetic ion beam that sputters atoms, clusters or large molecules (up to ~10,000 amu) off the surface. In TOF-SIMS, the ionised fraction of the sputtered secondary particles is detected directly. In laser SNMS, the sputtering and ionization processes are decoupled, by applying a laser beam to ionize the neutral particles sputtered from the sample surface, which in many situations has been shown to result in a significant improvement in detection sensitivity compared with TOF-SIMS. A number of different laser systems are available for photoionization; depending on the analytical objective, these can be used for various postionization schemes such as resonant and non-resonant multiphoton ionisation and single-photon ionisation. Both methods use time-of-flight mass spectrometers for the mass analysis, which due to a unique combination of high mass resolution, wide accessible mass range and transmission are ideally suited for sensitive analysis of biomolecules.
The potential of TOF-SIMS / laser-SNMS for chemical imaging in biological cells and tissue has long been recognized and a substantial amount of research has been invested in this area. However, the amount of new information obtained until quite recently was strongly limited, primarily due to (i) insufficient sensitivity (secondary ion yield), resulting in images with very weak signal intensities and (ii) extensive fragmentation, preventing a precise identification of large organic molecules. Several recent developments have dramatically advanced the state-of-the-art of imaging mass spectrometry with TOF-SIMS and laser-SNMS. Breakthroughs in ion beam technology, more specifically the development of ion sources producing cluster ions such as Au3+ Bin+ and C60+, have lead to substantial improvements in detection yields. The lateral resolution for these sources is currently 100/200 nm and 2-4 µm for Bin+ / Au3+ and C60+, respectively. Proof of principle for local analysis at lateral resolutions of a few 100 nm and absolute detection limits in the 10-20 mole range (~10 000 molecules) has been demonstrated with TOF-SIMS for selected organic molecules (asthma drug salbutamol). For biological samples, however, the complex morphological and molecular structure results in a substantial decrease in yield, and images with lateral resolution better than 0.5-1 µm have yet to be demonstrated. The number of biomolecules for which such images have been demonstrated is less than 10.
Another important ingredient for advancing the state-of-the art of these techniques is the development and increasing use of various statistical methods for analysis of mass spectra and images, which have lead to dramatically improved possibilities to identify different molecules in complex samples, such as biological ones. In addition, the use of femtosecond lasers for the ionisation step in laser SNMS has recently been shown to radically reduce the fragmentation of large molecules, which otherwise is a limiting factor for the biomolecule-identification capability of this technique.
Based on the recent progress in TOF-SIMS / laser SNMS technology, we are today at a stage where it will be possible to develop a new methodology for the analysis of biological samples. There is currently no technique available for parallel mapping of arbitrary substances in biological samples with sub-cellular spatial resolution. The aim of NANOBIOMAPS is to develop and promote the use of such a method.
Project content
NANOBIOMAPS will achieve its objectives by pursuing research end development in the following four areas:
· Instrument development: A new ion column, optimized for use with Bin+ clusters as the primary ions, will be developed. The instrumentation will provide imaging capability at a lateral resolution <50 nm. The development and optimization of tunable femtosecond laser (190 nm-2400 nm) is also included in the project.
· New or improved preparation methods for cell and tissue samples: One major challenge in establishing the TOF-SIMS and laser-SNMS techniques as methods for analysis of biological samples, is the fact that the analysis is done under ultra-high vacuum conditions, while biological systems naturally are wet. In order to transfer the biological samples from their native state to vacuum, the NANOBIOMAPS project will develop techniques for freezing hydrated samples, freeze drying, cryofracturing, and imprinting techniques. The latter two methods will also be explored as sample preparation methods for 3D-mapping at depth resolutions in the 10 nm 10 mm range. In addition, different sample preparation approaches towards maximizing secondary ion yields and detection limits will be attempted.
· Optimization of measurement conditions and analysis procedures: Maximizing the results obtained from TOF-SIMS and laser-SNMS analyses requires careful selection of analysis conditions and instrument parameters. Using well defined and characterized biological model systems, the NANOBIOMAPS project will investigate the role of different analysis conditions such as primary ion species and energy and sample temperature, to establish optimal analysis conditions for different types of biological samples.
· Data interpretation methods: TOF-SIMS and laser-SNMS produces huge amounts of data, which can be difficult to overview. This is especially the case with biological samples. To maximize the use of spectral data and ion images, different methods for multivariate statistical analysis for spectrum and image analysis will be developed and applied in the project. Data obtained in the project will also be collected in a database containing reference spectra for selected biological substances and samples.
Potential applications
Although not focused on applications, the NANOBIOMAPS project intends to explore different biological issues where the capabilities of TOF-SIMS and laser-SNMS can provide valuable new information. Among these, the following can be mentioned:
· Lipids and lipid membranes: Lipids act as the main building blocks of cell membranes, and also play an active role in several cell processes. The ability to identify and localize lipids at a sub-cellular resolution may provide new insights to e.g., lipid metabolism and the structure and dynamics of cell membranes. Particular issues that are planned to be investigate include imaging of lipid domains (lipid rafts), the composition of philopodia, asymmetry of inner and outer leaflets of the plasma membrane, and artificial supported membranes.
· Neurodegenerative diseases: Knowledge about the chemical and morphological changes occurring in cells and tissues as a result of different diseases, such as cancers, neurodegenerative diseases (e.g., Alzheimer’s disease) or immunological disorders, can give important insights into their cause(s) and development and also lead to the discovery of new early markers of the disease. A specific issue under investigation is the brain lesions (plaques) associated with Alzheimer’s disease.
· Cell behaviour at artificial surfaces: Understanding and controlling cell behaviour (adhesion, activation, differentiation) at artificial substrates is a key issue in a number of situations, such as biocompatibility of medical implants, tissue engineering using stem cells anchored to substrates and scaffolds, and emerging applications based on cells for drug screening or sensing of toxins. Cell-surface interaction is influenced and can be controlled by topography and chemical variations down to the nanometre scale. Imaging mass spectrometry offers new possibilities for investigating the chemical aspects of the cell substrate interface.
· Biosensors, protein arrays and gene chips: There is intense methods development around biosensors and analysis/diagnostic microarrays based on surfaces designed for selectively binding specific biological analytes, such as proteins and peptide or DNA sequences. As these devices are becoming increasingly miniaturized, imaging mass spectrometry may become a valuable tool both in their development and as a chemically specific reading method with high sensitivity and lateral resolution.
· Other applications: The methods and competence that will be developed in NANOBIOMAPS is anticipated to find uses also in areas outside the biomedical one. Specific examples that can be mentioned are food technology, and environmental sciences.
The partners behind the NANOBIOMAPS consortium encourage biological and medical researchers to contact us for a discussion about other biological samples or issues where we can contribute to new information.
NANOBIOMAPS
Project description