Overview of the results:
(1) We have developed a complete spectroscopy and microscopy system for performing nonlinear optical (NLO) imaging in a multimodal approach, combining stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence and second-harmonic generation. This instrument brings a very rich biochemical information on the sample under study. Up to now, NLO microscopes were based on complex and costly laser sources and detection systems, thus hindering its mainstream use in biology. We have greatly simplified the excitation scheme and reduced the costs and maintenance of the experimental setup, employing a fiber laser source. This instrument allowed us to study lipid accumulation in human breast cancer cells injured by iron depletors, an important step in the research about the mechanisms of tumours.
(2) We demonstrated a new approach to broadband SRS spectroscopy and microscopy, based on time-domain Fourier-transform (FT) spectroscopy, blending the very high sensitivity of single-channel lock-in balanced detection with the spectral resolution afforded by FT spectroscopy. Our novel approach paves the way to high-speed chemical identification of biomolecules via their broadband coherent Raman response. Then, we extended these results, realizing two related detection schemes capable of measuring the complex third-order nonlinear vibrational susceptibility of molecules, thanks to the use of an intrinsically phase-coherent local oscillator. This in turn provides access to the vibrational phase, which allows one to distinguish overlapping species in congested spectra and is more robust with respect to noise.
(3) We demonstrated a novel approach to broadband SRS, based on Photonic time stretch (PTS) spectroscopy. PTS is a powerful technique for single-channel measurement of optical spectra at high repetition rates, up to tens of MHz.
(4) We demonstrated a novel approach to broadband SRS, based on a home-built compact multi-channel lock-in amplifier with 32 channels. We have performed extensive tests and confirmed that it performs equally well as commercial high-frequency lock-in amplifiers, which are much more expensive and large in volume and cannot work with more than one channel. This is a tremendous step forward in broadband SRS.
(5) We have employed artificial intelligence to provide an extra dimension to CARS spectroscopy and microscopy. Deep learning algorithms enable to remove the unwanted non-resonant background and thus retrieve the pure vibrational Raman signatures of molecules under study.
(6) We introduced In-line Balanced Detection (IBD), a novel configuration for SRS microscopy to increase the signal to noise ratio in a very simple way.
(7) We introduced a high-sensitivity broadband (SRS) setup featuring wide spectral coverage and high-frequency resolution, using a single-channel lock-in amplifier connected to a single-pixel photodiode. The spectrum is scanned rapidly using a galvanometric mirror after the sample, obtaining hyperspectral data cubes.
Exploitation and dissemination:
This ERC project VIBRA dealt with a basic ground-braking research. Therefore, the dissemination of the results has focused mainly in scientific publications. We have published several posts on social media to advertise the results and the impact to the society. The developed techniques constitute a fundamental step towards future application of the coherent Raman scattering spectroscopy and microscopy technique in fields such as biological research and clinical practice. The technology readiness level (TRL) has been pushed from the initial TRL1 to the actual TRL3-4. The next step will be to exploit the results and to realize a CRS microscope at high TRL for commercial exploitation. The potential market is very big and such an instrument could have a revolutionary impact in many fields such as biological and medical research, to image cells and tissue and gain an unprecedented level of bio-molecular information to study several diseases. This has been the topic of a joint H2020 project proposal recently submitted, coordinated by Prof. Dario Polli, which could take the results of VIBRA to the next level and realize a broadband CRS microscope in collaboration with 9 other partners in Europe, including SMEs to bring the technology to the market and biomedical doctors and researchers to validate the results in three case studies.
