In the past 50 years the progress of life sciences has been propelled by the understanding of biological mechanisms at a molecular level. The knowledge of the structure of several supra-molecular systems and the success of the genome sequencing efforts have been important milestones of a challenging pathway whose next objective is the understanding of the functions of bio-molecules within cells. To this purpose optical microscopy represents an extremely powerful investigation tool thanks to its ability of visualizing morphological details in cells and tissues on the sub-micrometer scale. It provides a much higher spatial resolution compared to magnetic resonance imaging, and at the same time it does not require the sample to be fixed, as in electron microscopy. Fluorescence microscopy using exogenous markers (such as dyes or semiconductor quantum dots) offers a superb sensitivity, down to the single molecule limit. Moreover, the discovery of genetically programmable endogenous fluorescent markers, such as the Green Fluorescent Protein, has strongly broadened its traditional application field.
However, the addition of fluorescent markers can hardly be implemented within certain cells or tissues, or it can give strong perturbation to the investigated system. This is particularly true for small molecules, such as signaling peptides, neurotransmitters, metabolites and drugs, for which the dimension of the marker is comparable to or even bigger than that of the molecule itself, so that it heavily interferes with its biological function. In addition, in many clinical applications involving live tissue imaging, staining with fluorophores is not possible or not desired. For these reasons, there is a great need for intrinsic, label-free imaging methods that do not require the addition of any fluorescent molecule. (fig.1)
Every component of a biological specimen (cell or tissue) is characterized by a vibrational spectrum that reflects its molecular structure and provides an endogenous and chemically specific signature that can be exploited for its identification (see Fig. 1). Vibrational absorption microscopy uses mid-infrared light (λ = 3-10 μm), directly resonant with a vibrational transition of the molecule; however, long wavelengths result in low spatial resolution and limited penetration depth due to water absorption. Spontaneous Raman microscopy detects the inelastically scattered light that is energetically downshifted by the characteristic vibrational frequency of a molecule (Stokes light), according to the scheme reported in Fig. 2(a). Since it uses visible or near-infrared light, it provides much higher penetration depth and spatial resolution. However, due to the very low cross section of the spontaneous Raman process (about 10 to 12 orders of magnitude lower than the absorption cross section), the generated signals are very weak, thus requiring high excitation powers and long integration times, preventing real-time imaging of living tissues and the study of dynamical processes.
A significant step forward in the field of vibrational microscopy is enabled by Coherent Raman Scattering (CRS) techniques, making use of two synchronized trains of laser pulses at frequencies ωp (Pump frequency) and ωS (Stokes frequency) providing resonant excitation of the Raman transition. CRS techniques exploit the third-order nonlinear optical response of the sample by setting up and detecting a vibrational coherence within the ensemble of molecules inside the laser focus. When the difference between Pump and Stokes frequencies matches a characteristic vibrational frequency Ω, i.e. ωp - ωS = Ω, then all the molecules in the focal volume are made to vibrate in phase; this vibrational coherence enhances the Raman response by many orders of magnitude with respect to the incoherent spontaneous Raman process. The two most widely employed CRS techniques are Coherent Antistokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS). In the CARS process (Fig. 2 (c)) the vibrational coherence is read by a further interaction with the pump beam, generating a coherent radiation at the anti-Stokes frequency ωaS = ωp + Ω; in SRS (Fig. 2(b)) the coherent interaction with the sample induces stimulated emission from a virtual state of the sample to the investigated vibrational state, resulting in a Stokes-field amplification (Stimulated Raman Gain, SRG) and in a simultaneous pump-field attenuation (Stimulated Raman Loss, SRL).
In summary, CRS microscopy provides the following advantages:
- In comparison with fluorescence microscopy it is label-free, i.e. it does not require fluorophores, allowing the study of unaltered cells and tissues;
- It typically works out of resonance, i.e. without population transfer into electronic excited molecular states, thus minimizing photobleaching and damage to biological samples;
- Since CRS exploits a coherent superposition of the vibrational responses from the excited oscillators, it is considerably more sensitive than spontaneous Raman microscopy, allowing extremely higher imaging speeds, up to the video rate;
- Being a nonlinear microscopy techniques, with the signal generation confined to the focal volume, it exhibits a three-dimensional sectioning capability similar to that of multiphoton fluorescence microscopy;
- The use of near-infrared excitation (700-1200 nm) has the advantage of a high penetration depth, which allows imaging through thick tissues, and a low phototoxicity, minimizing multi-photon absorption induced damage.
The goal of this research activity is the development of a fiber-format laser system featuring an unprecedented degree of compactness, robustness, cost-effectiveness and transportability, based on Er-fiber technologies. The developed system will give easy access, to the user’s choice, either to an SRS approach equipped with an ultra-low-noise detection chain or to a CARS approach employing an NRB suppression technique, so as to offer the highest level of sensitivity and chemical selectivity. Several applications are under study, ranging from biomedical imaging to the study of nanomaterials. The European Research Council (ERC) has granted a 1.8M€ project to Politecnico di Milano to develop an innovative broadband coherent Raman scattering microscope. Further information can be found at: www.vibra.polimi.it