At NANOTEC several high resolution techniques are available for fine imaging of biomolecules, living cells and tissue. Mainly we focus on engineering of high-resolution molecular and semiconductor-based tools for fine fluorescence imaging, scanning probe-based microscopy and spectroscopy, and X-ray optics and X-ray imaging.
Fluorescent Tools for Bioimaging of Cells and Tissues
We prepare various kinds of fluorescent imaging tools for in vitro and in vivo imaging, mainly of cells and tissues. This class of labelling agents include organic thiophene fluorophores, and semiconducting quantum dots (QDs) or rods (QRs). Our thiophene and QD-based reagents show strong luminescence that is independent of the microenvironment, may be designed to work in the optical window of biomatter (600 – 900 nm range), display rapid internalization and excellent retention inside cells, and appear to be non toxic at least in short-term experiments. In addition, in the case of inorganic nanoparticles large surface/volume ratios enable biofunctionalization of the surface and binding of functional moieties for specific targeting studies. Another set of imaging tools includes fluorescent ratiometric capsule-based sensors, which have fluorescent probes embedded in their cavities, allowing to overcome some of the main limitations of freely injected probes (e.g. intracellular sequestration or inactivation, non-specific binding, toxicity). These sensor platforms are suitable for long-term intracellular and extracellular sensing of multiple chemical parameters (ions and metabolites) in living and metabolizing cells, including tumor cells or cells exposed to candidate drugs.
Nanoscale Characterization of Biomaterials and Nanostructured Surfaces
We employ state-of-the-art microscopy techniques (e.g., Scanning Probe Microscopies like Atomic Force Microscopy (AFM) and Scanning Tunneling Microscopy (STM), Scanning Electron Microscopy, Confocal Laser Microscopy) for studying the morphological and chemico-physical properties of nano(bio)materials and nanostructured surfaces, as well as to gain a full understanding of the correlation between the fabricated structures and the resulting functionality. By means of STM, AFM and Scanning Force-Spectroscopy analyses, we have extensively characterized the morphological, mechanical and conductive properties of solid-state protein films and self-assembled peptide fibrils to be used as bridges in hybrid molecular junctions. Finally, we synthesize and characterize biomimetic and biocompatible surfaces with stiffness modulation and/or a biopolymeric coating, for eliciting controlled cell self-patterning, proliferation and differentiation with important implications in regenerative medicine.
The team in Rome has a large experience in X-ray optics and X-ray imaging. In recent years the team is exploiting this competence for the biomedical imaging of neurodegenerative diseases. In particular, faults in vascular (VN) and neuronal networks (NN) of the spinal cord are associated with serious neurodegenerative pathologies. Because of inadequate investigation tools, the knowledge of the fine structure of VN and NN is fragmentary, which represents a crucial problem. Conventional 2D imaging yields incomplete spatial coverage leading to possible data misinterpretation, whereas standard 3D computed tomography imaging achieves insufficient resolution and contrast. X-ray high-resolution phase-contrast tomography (XPCT) allows the simultaneous visualization of three-dimensional VN and NN of ex-vivo mouse spinal cord at scales spanning from millimeters to hundreds of nanometers, withoutcontrast agents, or sectioning or destructive sample-preparation. We image both the 3D distribution of micro-capillary network and the micrometric nerve fibres, axon-bundles and neuron soma. The high quality of the images obtained allows a quantitative study, enabling the extraction of relevant biomedical information.
Our approach is well suitable for pre-clinical investigations of neurodegenerative pathologies and spinal-cord-injuries. In this framework, we used XPCT to study degenerations in vascular and neuronal networks of the spinal cord affected by experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Preclinical studies have demonstrated that mesenchymal stem cells (MSC) therapy ameliorates EAE. By means of XPCT, we investigated 3D sub-micron damage in the VN and NN in EAE with and without MSC treatment. We have identified a deficit in the Vascular-Network, including capillaries, never detected before, and which is reduced through MSC treatment. Similarly, the attack to myelin and neurons also appears to be reduced by MSC administration.
This study paves the way to advanced study of immune-mediated CNS diseases, and to the accurate monitoring of the effectiveness of treatment.
- L. L. del Mercato, F. Guerra, G. Lazzari, C. Nobile, C. Bucci, R. Rinaldi. Biocompatible multilayer capsules engineered with a graphene oxide derivative: synthesis, characterization and cellular uptake. Nanoscale. 8, 7501-7512 (2016). ISSN: 2040-3372; doi: 10.1039/C5NR07665J.
- I. Bukreeva, A. Mittone, A. Bravin, G. Festa, M. Alessandrelli, P. Coan, V. Formoso, R.G. Agostino, M. Giocondo, F. Ciuchi, M. Fratini, L. Massimi, A. Lamarra, C. Andreani, R. Bartolino, G. Gigli, G. Ranocchia, A. Cedola. Virtual unrolling and deciphering of Herculaneum papyri by X-ray phase-contrast tomography, Scientific Reports 6, 27227 (2016). ISSN: 2045-2322; doi: 10.1038/srep27227
- L. L. del Mercato, M. M. Ferraro, F. Baldassarre, S. Mancarella, V. Greco, R. Rinaldi, S. Leporatti. Biological Applications of LbL Multilayer Capsules: From Drug Delivery to Sensing. Advances in Colloids and Interface Science, 207, 139–154, (2014). ISSN: 0001-8686; doi: 10.1016/j.cis.2014.02.014.
- De Luca, M.M. Ferraro, R. Hartmann, P. Pilar Rivera-Gil, A. Klingl, M. Nazarenus, A. Ramirez, W.J. Parak, C. Bucci, R. Rinaldi, L.L. del Mercato. Advances in Use of Capsule-Based Fluorescent Sensors for Measuring Acidification of Endocytic Compartments in Cells with Altered Expression of V-ATPase Subunit V1G1. ACS Appl. Mater. Interfaces, 7, 15052−15060 (2015). ISSN 1944-8244; doi: 10.1021/acsami.5b04375.
- L. L. del Mercato, M. Moffa, R. Rinaldi, D. Pisignano. Ratiometric Organic Fibers for Localized and Reversible Ion Sensing with Micrometer-Scale Spatial Resolution. Small, 11, 6417-6424 (2015). ISSN: 1613-6810; doi: 10.1002/smll.201502171.
- M. Fratini, G. Bukreeva, F. Campi, G. Brun, P. Tromba, D. Modregger, G. Bucci, R. Battaglia, M. Spanò, H. Mastrogiacomo, F. Requardt, A. Giove, A. Bravin and A. Cedola. Simultaneous submicrometric 3D imaging of the micro-vascular network and the neuronal system in a mouse spinal cord. Scientific Reports 5, 8514 (2015). ISSN: 2045-2322; doi: 10.1038/srep08514.
- M. Fratini, I. Bukreeva, G. Campi, F. Brun, G. Tromba, P. Modregger, D. Bucci, G. Battaglia, R. Spanò, M. Mastrogiacomo, H. Requardt, F. Giove, A. Bravin, A. Cedola Simultaneous submicrometric 3D imaging of the micro-vascular network and the neuronal system in a mouse spinal cord, Scientific Reports 5, 8514 (2015). ISSN: 2045-2322; doi: 10.1038/srep10771
- M. Fratini, I. Bukreeva, G. Campi, R. Spano’, M. Mastrogiacomo, F. Brun, G. Tromba, F. Giove, A. Cedola. Study of the vascular network in the spinal cord using advanced techniques Journal of Tissue Engineering and Regenerative Medicine 8, 192 (2014). ISSN: 1932-6254.
- Di Maria, I.E. Palamà, M. Baroncini, A. Barbieri, R. Bizzarri, G. Gigli, G. Barbarella, Live Cell Cytoplasm Staining and Selective Labeling of Intracellular Proteins by Non-toxic Cell-permeant Thiophene Fluorophores. Organic & Biomolecular Chemistry, 12: 1603-1610 (2014). ISSN: 1477-0520; doi: 10.1039/C3OB41982G
Other selected publications
- L.L. del Mercato, P.P. Pompa, G. Maruccio, A. Della Torre, S. Sabella, A.M. Tamburro, R. Cingolani, R. Rinaldi, Charge transport and intrinsic fluorescence in amyloid-like fibrils. Proceedings of the National Academy of Sciences (PNAS), 46, 18019-18024 (2007). ISSN: 0027-8424; doi: 10.1073/pnas.0802398105
- L.L. del Mercato, G. Maruccio, P.P. Pompa, B. Bochicchio, A.M. Tamburro, R. Cingolani, R. Rinaldi, Amyloid-like fibrils in elastin-related polypeptides: structural characterization and elastic properties. Biomacromolecules, 9, 796–803 (2008). ISSN: 1525-7797; doi: 10.1021/bm7010104
- A. Zacheo, A. Quarta, A. Mangoni, P.P. Pompa, R. Mastria, M.C. Capogrossi, R. Rinaldi, T. Pellegrino. CdSe/CdS Semiconductor Quantum Rods as Robust Fluorescent Probes for Paraffin-Embedded Tissue Imaging. IEEE Transactions on Nanobiosciences, 10, 3, 209-215 (2011). ISSN: 1536-1241; doi: 10.1109/TNB.2011.2166404.
- I.E. Palamà, F. Di Maria, I. Viola, E. Fabiano, G. Gigli, C. Bettini, G. Barbarella. Live-cell-permeant thiophene fluorophores and cell-mediated formation of fluorescent fibrils. Journal of the American Chemical Society (JACS), 133, 17777–17785 (2011). ISSN: 0002-7863; doi: 10.1021/la2065522.
- L.L. del Mercato, A.Z. Abbasi, M. Ochs, W.J. Parak. Synthesis and characterization of ratiometric ion-sensitive polyelectrolyte capsules. Small, 7, 351-363 (2011). ISSN: 1613-6810; doi: 10.1002/smll.201001144.
- A. Quarta, A. Curcio, H. Kakwere, T. Pellegrino. Polymer Coated inorganic nanoparticles: tailoring the nanocrystal surface for designing nanoprobes with biological implication. Nanoscale, 4, 11, 3319-333 (2012). ISSN: 2040-3364; doi: 10.1039/c2nr30271c.
- L.L. del Mercato, A.Z. Abbasi, M. Ochs, W.J. Parak. Multiplexed Sensing of Ions with Barcoded Polyelectrolyte Capsules. ACS Nano, 5, 9668–9674 (2011). ISSN: 1936-0851; doi: 10.1021/nn203344w
- I.E. Palamà, A.M.L. Coluccia, G. Gigli, M. Riehle. Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials. Integrative Biology, 4, 1299-1309 (2012). ISSN: 1757-9694; doi: 10.1039/c2ib20133j.
- I.E. Palamà, S. D’Amone, A.M.L. Coluccia, M. Biasiucci, G. Gigli. Cell self-patterning on uniform PDMS-surface with controlled mechanical cues. Integrative Biology, 4, 228-236 (2012). ISSN: 1757-9694; doi: 10.1039/c2ib00116k.
- I.E. Palamà, S. D’Amone, A.M.L. Coluccia, G. Gigli. Micropatterned polyelectrolyte multilayer films promote alignment and myogenic differentiation of C2C12 cells in standard growth medium. Biotechnology & Bioengineering, 110, 586-596 (2013). ISSN: 0006-3592; doi: 10.1002/bit.24626.