Raman spectroscopy is currently employed as one of the most powerful tools for the investigation of the nature of matter. It employs a focused laser beam that selects a specific portion of the sample (when used in conjunction with a microscope, the selected area can be as small as 1 micron squared). The light reflected and scattered by the sample is analyzed by a spectrometer, and the few photons (light waves) that have changed their energy (frequency) during interaction with the sample are identified in the so-called Raman spectrum. Peaks in the Raman spectrum reveal specific molecules, or details of the bandstructure of solids, or the conformation of proteins. Raman spectroscopy is already well represented at Sapienza. Here we propose to extend the pre-existing instrument selection by coupling to a Raman spectrometer employing an infrared laser (wavelength of 1064 nm) a purposely designed microscope now commercially available.
Raman spectroscopy has recently played a key role in the study of graphene. Therein the resonant nature of the interaction between the excitation laser light and the electrons modifies the frequency and the lineshape of the Raman peaks. The use of infrared lasers instead of visible lasers enables the Raman investigation of a new range of electron energies and of new portions of the unique band structure of graphene, much closer to the Dirac point, whose discovery lead to the Nobel Prize in Physics in 2010.
Raman spectroscopy is a purely photonic technique hence contact-less, ideal for investigating artworks and biosystems, however, the focused visible laser light can induce a strong temperature increase or even permanent damage in the sample. Infrared light is much less strongly absorbed than visible light by e.g. cellulose and pigments, or by proteins and fluorescent chromophores. An infrared Raman spectrometer would permit Raman studies of precious artworks and of light-sensitive biosystems with strong levels of natural fluorescence.
The acquisition of a FT Raman micro-spectrometer, that would complement the spectroscopic techniques already in use in our departments, will allow the opening of novel research lines and the implementation of those already successful. The use of FT Raman in conjunction with a microscope will enable measurements otherwise prevented and in particular we envision to have results in the following topics:
Expected innovation in A1: The use of high aperture optics at the sample collection site will enable for the first time to collect the Raman signal, with long wavelength excitation, arising from a single layer of graphene. Recently we have obtained indication that Raman spectra obtained with excitation laser wavelength of 1 micron and longer may provide the best possible test for the most advanced electron-phonon scattering theories. Indeed, as graphene devices are becoming more common, the material quality is still the limiting factor for electronic device performance. Phonon branches involved in scattering processes could be 1) the instrinsic Raman-active optical phonons and 2) the acoustic phonons of graphene, or 3) the optical phonons of the substrate and/or encapsulating layers adhering to graphene. These interactions can be modeled with very high precision starting from first-principle atomistic calculations based on the density functional theory of the Raman spectra of graphene.
Expected innovation in A2: This Raman setup, with the refurbishment and the coupling of the microscope will enable spectroscopic measurements at ultra-high pressure, as the fluorescence of the diamonds will be circumvented via the long-wavelength excitation. The use of the microscope, together with the optical coupling between the multiRAM and the microscope will guarantee the optimal focusing of the laser and collecting of the Raman signal. Ultrahigh pressure measurements can be performed only on extremely small samples (lateral size of the order of few microns), therefore we expect to have a poor signal to noise ratio. In this perspective, the impressive stability of the multiRAM Ge detector will be a key ingredient to success.
Expected innovation in B: The use of a Raman microscope with excitation at IR wavelengths has the potential to obtain non-destructive diagnostic results not only on manuscripts but also on cultural heritage material in general, such as . The information obtained can be used to increase the historical-archaeological knowledge of the manufacturing technology of a product, the exclusive nature of the materials used and their state of degradation for a specific and timely conservation definition. It is noteworthy that with the implementation of such a microscope, which is not available in the Lazio region, one would increase the range of the instrumentation available for the ¿Distretto dei beni culturali del Lazio¿, of whom Sapienza is already partner. The development of spectroscopic methods that could be non-destructive for cultural heritage materials has opened up the possibility of carrying out novel diagnostic analysis. The possible results bear not only on aspects of restoration and conservation, but also on the authentication and dating of artwork. The work will be conducted as a first step on illuminated manuscripts with the aim of a comprehensive characterization of the used pigments, while searching for signatures of specific artistic techniques or of old restorations performed on the original drawing.
Expected innovation in C: The use of FT-Raman microscopy will be done in conjunction with microfluidic cells, to study the subtle protein modifications due to interaction with external stimuli, receptors and in the presence of different loaded compounds while kept in a physiological solution whose concentration of chemical species can be directly controlled and modified with no need of removing the sample from the focal plane of the microscope. Also, the thickness of the aqueous layer will be in this way kept constant in the optical path, allowing for concentration-dependent spectroscopy of proteins.
As a first step we will focus on ferritin-based systems and on channelrhodopsin, a transmembrane protein that can directly function as ion channel or pump by means of visible light absorption. This protein in particular is of great interest for optogenetics, but is extremely sensitive its environment (hydration among all). As the FT-Raman signal, due to its longer wavelength excitation, will be dominated by the chromophore normal vibration modes, we will perform FT-Raman imaging (with a lateral resolution of about 500 nm) for both light-adapted and dark-adapted state of the channelrhodopsin and compare the results with Infrared absorption imaging. This will allow for the first comprehensive description of the light-induced process as both the chromophore and the consequent conformational change of the protein are probed in the same sample.