The Center for Nanotechnology Innovation (CNI) is an interdisciplinary R&D center dedicated to the investigation and exploitation of phenomena at the nanoscale. The center is embedded in the NEST laboratories of the Scuola Normale Superiore in Pisa and is part of the network of the Istituto Italiano di Tecnologia (IIT) whose headquarters are in Genova. CNI research lines span from molecular medicine to new nano-diagnostic, from 2D materials engineering and design to electron crystallography and new advanced microscopy methods.
Within the center which is coordinated by Mauro Gemmi, Camilla Coletti works also as indipendent PI developing the 2D Materials Engineering research line.
The term electron crystallography joins all the crystallographic studies carried out using electrons as a probe either in diffraction or in imaging. The ZEISS Libra 120 transmission electron microscope at the CNI@NEST has been configured as an electron diffraction station for electron crystallography studies.
Electron diffraction tomography: this research activity is focused on the precession assisted electron diffraction tomography (PEDT) method for its application to very beam sensitive materials as organics, pharmaceutical and proteins. On “hard” inorganic materials our electron crystallography station is already able to furnish diffraction data suitable for both structure solution and refinements on crystal as small as few hundreds of nanometers (dynamical refinement). We are actually developing a completely automatic data collection procedure (Fast EDT) that, coupled with a new direct electron diffraction camera (MEDIPIX), will allow fast data collections in less than 1 minutes drastically reducing the dose suffered by the sample. We expect in the very near future to be able to collect in this way data on vitrified samples containing protein nanocrystals which will be suitable for solving the protein crystal structure.
Nano texture analysis: this activity is based on the ASTAR system for collecting phase and orientation mapping. We are trying to increase the resolution and the data collection speed by using the MEDIPIX detector as recording device. Exploiting its sensitivity we expect to bring the resolution down to the minimum beam size available of 1.4 nm. Possible applications: polytypism at the nanoscale of pharmaceutical, evaluation of the nanocrystalline amorphous ratio in beam sensitive samples.
Correlative TEM Micro-CT
The lab is developing a new correlation method between X-Ray tomography (Micro-CT, in collaboration with SYRMEP beamline at Elettra - Sincrotrone Trieste) and transmission electron microscopy. The tomographic 3D reconstruction of whole organs and/or tissue already embedded and fixed for TEM analysis allows the identification of region of interest (usually pathological hallmarks) with the intrinsic resolution of few microns characteristic of Micro-CT. The sample can then be cut with the ultra-microtome just across the identified region of interest for obtaining a this section suitable for TEM imaging, avoiding the time-consuming method of serial sectioning and the loss of regions that could be important in the characterization of particular models of unpredictable pathologies in which ROIs are randomly localize within the sample.
Coherent anti-Stokes Raman spectroscopy
Coherent anti-Stokes Raman Scattering (CARS) microscopy permits label-free imaging with excellent chemical selectivity and high spatial resolution. It is a very promising tool for a broad range of research areas, from biophysics studies at sub-cellular level to material science at the nanoscale.
The frequency difference of "pump" and "Stokes" photons is selected to resonantly excite the target vibration mode (ωvib). This excitation is probed by a third photon that induces a coherent emission of anti-Stokes radiation. Signal intensities several orders of magnitude stronger than spontaneous Raman can be achieved thanks to the coherent build-up of the light field and the use of pulsed lasers.
At the CNI we developed a large-field-of-view polarization-resolved CARS microscope and devised a technique, rotating-polarization CARS (RP-CARS), that allows determining the spatial anisotropy of selected chemical bonds and their average orientation. RP-CARS is the technique of choice to image myelinated axons because of the strong CARS signal from the abundant CH2 bonds.
We have shown that the degree of anisotropy of the CH2 bonds in myelin sheaths (quantified with a pixel-based numerical indicator) presents a significant correlation with their health status in a chemical model of demyelination, paving the way for clinical applications of RP-CARS in the field of neurodegenerative disorders.
Real-time 4D molecular analysis in dynamic subcellular nanostructures
In many naturally occurring circumstances single-molecules are exerting their functions as part of a nanoscopic system that is continuously and rapidly changing position and shape in time and space. No method exists today that has the capability to fruitfully investigate single-molecule behavior in these conditions, in a 3D environment. The CNI@NEST aims at addressing this challenging task by the development of new fluorescence-based imaging and analysis methods with high spatiotemporal resolution. As strategic platform, we propose to focus an excitation beam in a periodic orbit around the (fluorescently-labeled) nanostructure of interest. The resulting signal will be used as feedback to localize the nanostructure position with an unprecedented combination of spatial (~10 nm) and temporal (~1000 Hz frequency response) resolution. Concomitantly, we are developing novel analytical tools based on spatiotemporal fluorescence correlation spectroscopy (FCS) that provide conceptually the same physical quantities of classical single particle tracking (SPT) techniques but using small, even dim, molecular labels (i.e. with minimal perturbation of the sample), with no need for interpretative models. Of particular note, the ability of these approaches to resolve average molecular dynamic properties well below the limit imposed by diffraction. By adapting and combining this toolbox of analytical approaches along the orbit, single-molecule investigations will become accessible on a moving reference system, for the first time. We believe that this strategy has the potential to develop a flexible, nanotechnology-based, multi-functional approach capable of addressing any high-impact biological question that involves single-molecule behavior on dynamic nanostructures, thus pushing ahead the frontier of current knowledge of living matter.
Nano Bio Design
The object of this research is the development of innovative Nano-architectures for medicine and catalysis. One of the major concerns regarding the clinical translation of metal nanoparticles is related to the question of persistence in organisms. The dilemma in the choice of particle size needed for clinical applications versus efficient body clearance has created a serious conflict in nanotechnology. In order to overcome these issues, our approach is aimed in designing inorganic disassembling Nano-architectures for cancer theranostics. The functionalizable hollow Nano capsules we introduced can maintain the intriguing features of metal nanostructures and are biodegraded in cellular environment to their components. Thanks to this approach, the issue of accumulation is potentially overcome due to the renal clearance of the building blocks. The possibility to modify the composition and the internal/external functionalization of the Nano capsules make them promising Nano tools for therapeutic and imaging applications, among which drug delivery, chemo/radio-therapy, photoacoustic and magnetic resonance.
Metal nanostructures have also demonstrated a wide range of applicability in catalysis, but the need of a surface coating reduces their efficiency. In order to fully exploit their potential, we designed and developed “naked” metal nanoparticles hold in and protected by permeable silica Nano capsules. Our efforts are in particular focused on air-pollutant removal (VOCs and NOx) and hydrogen storage.
Magnetic Resonance Imaging is a powerful diagnostic technique, widely applied in the clinic and in pre-clinical research, offering several advantages such as non-invasive imaging, deep tissue penetration, and superior spatial resolution. Magnetic contrast agents are routinely used to enhance image contrast, and help discriminate between different tissues. Magnetic NanoParticles (MNPs) have also been proposed as MRI contrast agents thanks to their ability to increase the transverse relaxation rate (R2) and bring negative contrast. Additionally, MNPs have been applied extensively in drug delivery and therapy (including chemotherapy) when conjugated with drugs, and in targeted molecular and biomolecular imaging. The development of targeted MNPs allows reduction of doses and selective delivery to malignant tissues. Objective of this project is the development of novel probes for Magnetic Resonance Imaging applications.
Specifically, we aim to:
- develop MR imaging MNPs capable of reporting quantitatively on the cellular microenvironment;
- design targeted nanoprobes with increased molecular specificity and sensitivity to target cellular and tissue compartments otherwise inaccessible to conventional contrast agents;
- develop novel contrast agents able to perform multi-modal imaging by combination of MRI with other diagnostic techniques, such as optical imaging or PET.
Laura Marchetti is a post-doc researcher with a solid background in molecular and cellular biology and a strong attitude to interdisciplinary approaches. She is currently working at the interface between biophysics and cellular biology, by applying single-molecule fluorescence imaging approaches to investigate the membrane and intracellular dynamics of neurotrophins and their receptors. She has contributed to the development of means to: i) evaluate the changes of receptor membrane mobility induced by ligand binding in living cells; ii) label both neurotrophins and their receptors with controlled stoichiometry and with fluorophores suitable for single-molecule imaging purposes; iii) control the expression level of recombinant proteins expressed in mammalian cells using lentiviral vectors and inducible promoters. These molecular tools were and are still being used in a number of different projects that allowed her to collaborate with different research groups (NEST and BioSNS Labs of Scuola Normale Superiore in Pisa, EBRI Institute in Rome, Department of Molecular and Translational Medicine of Brescia Univeristy) and supervise the activity of several MSc and PhD students.
Designing protein molecules that self-assemble into complex bio architectures is an innovative goal of Nano biotechnology. Applications can range from design of bioactive 3D Nano biomaterials, to Nano Bio carriers and Nano biosensors, from bioelectronics to biomedicine. Nature has evolved already a large variety of complex biostructures at nanoscale for cell life. These arise mainly from assemblies of different proteins through non-covalent and highly specific interactions, such as viral capsids and microtubules. The aim of our research activity is to engineer and generate novel bioinspired (metallo)protein scaffolds that can self-assemble upon stimulation into desired well-ordered and stable multicomponent Nano-biostructures, such as bio molecular cages and crystals. This process is strongly driven by specific technological needs.
Primary research tools include approaches of protein engineering, overexpression of recombinant proteins in mammalian and bacterial cell systems, production scale-up, bio crystallization, and structural biophysics analysis (mainly x-ray crystallography, small angle x-ray scattering, neutron scattering, cryo-electron microscopy and tomography, surface plasmon resonance and isothermal titration calorimetry). We have access to major EU research platforms of synchrotron light source. Overall, these methods provide insights into protein structure and dynamics at the atomic level, and allow us to characterize the thermodynamics and kinetics of protein interactions, which are key parameters for efficient protein architecture design and hierarchical subunit assembly.
This research line focuses on the development of synthetic sequences for targeted delivery of therapeutic or diagnostic payloads in cells. We mainly focus on the rational engineering and optimization of known peptide/oligonucleotide aptamers by means of combined computational/experimental approaches, and on the development of new architectures able to perform targeted co-delivery of therapeutic agents. Compared to traditional small drugs, this strategy provides increased therapeutic activity with negligible off- target effect, thus allowing use of lower drug doses. Additionally, targeted co-delivery allows performing two therapeutic actions at the same time, e.g. cytotoxic activity of the drug and inhibition of natively constituted anti-apoptotic mechanisms of target cells.
These sequences are currently studied as homing agents for large nanostructures such as advanced seed-based, polymeric, and self-assembled nanoparticles.
- TEM Lab
- Synthesis Facilities
- Confocal Microscopy
- 2D materials Characterization
- 2D materials Synthesis
- Cars Microscopy
The CNI is embedded and shares its research facilities with the NEST lab: NEST facilities.