Label-free polarisation-resolved optical imaging of biological samples

Myelin is a biological structure present in all the gnathostomata. It is a highly- ordered structure, in which many lipid-enriched and densely compacted phospho- lipid bilayers are rolled up in a cylindrical symmetry around a subgroup of axons. The myelin sheath increases the electrical transverse resistance and reduces the ca- pacitance making the saltatory conduction of action potentials possible and therefore leading to a critically improved performance in terms of nervous impulse conduc- tion speeds and travel lengths. Myelin pathologies are a large group of neurological diseases that often result in death or disability. In order to investigate the main causes of myelin damage and its temporal progression many microscopy techniques are currently employed, such as electron microscopy and histochemistry or fluorescence imaging. However, electron microscopy and histochemistry imaging require complex sample prepara- tion and are therefore unsuitable for live imaging. Fluorescence imaging, as well as its derivatives, confocal and two-photon imaging, relies on the use of fluorescent probes to generate the image contrast but fluorophores and the associated sample processing, when applicable to living specimens, might nonetheless modify the bi- ological properties of the target molecule and perturb the whole biological process under investigation; moreover, fluorescent immunostaining still requires the fixation of the cells. Coherent anti-Stokes Raman Scattering (CARS) microscopy, on the other hand, is a powerful and innovative imaging modality that permits the study of liv- ing specimens with excellent chemical contrast and spatial resolution and without the confounding and often tedious use of chemical or biological probes. This is par- ticularly important in clinical settings, where the patient biopsy must be explanted in order to stain the tissue. In these cases it may be useful to resort to a set of label-free microscopy techniques. Among these, CARS microscopy is an ideal tool to investigate myelin morphology and structure, thanks to its abundance of CH2 bonds. The chemical selectivity of CARS microscopy is based on the properties of the contrast-generating CARS process. This is a nonlinear process in which the energy difference of a pair of incoming photons (“pump” and “Stokes”) matches the energy of one of the vibrational modes of a molecular bond of interest. This vibrational excited state is coherently probed by a third photon (“probe”) and anti-Stokes radi- ation is emitted. In this thesis I shall discuss the development of a multimodal nonlinear opti- cal setup implementing CARS microscopy together with general Four-Wave Mix- ing, Second Harmonic Generation and Sum Frequency Generation microscopies. Moreover, I shall present a novel polarisation-resolved imaging scheme based on the CARS process, which I named Rotating-Polarisation (RP) CARS microscopy and implemented in the same setup. This technique, using a freely-rotating pump-and- probe-beam-polarisation plane, exploits the CARS polarisation-dependent rules in order to probe the degree of anisotropy of the chemical-bond spatial orientations inside the excitation point-spread function and their average orientation, allowing at the same time the acquisition of large-field-of-view images with minimal polarisa- tion distortions. I shall show that RP-CARS is an ideal tool to investigate the highly- ordered structure of myelinated nervous fibres thanks to the strong anisotropy and symmetry properties of the myelin molecular architecture. I shall also demonstrate that this technique allows the fully label-free assessment of the myelin health status both in a chemical model of myelin damage (lysophos- phatidylcholine-exposed mouse nerve) and in a genetic model (twitcher mouse) of a human leukodystrophy (Krabbe disease) while giving useful insights into the pathogenic mechanisms underlying the demyelination process. I shall also discuss the promises of this technique for applications in optical tractography of the nerve fibres in the central nervous system and for the investigation of the effects of ageing on the peripheral nervous system. Moreover, I shall demonstrate by means of numer- ical simulations that RP-CARS microscopy is extremely robust against the presence of scatterers (such as lipid vesicles, commonly found in the peripheral nervous sys- tem). Finally, I shall discuss the results of the exploitation of my multimodal setup in a different area at the boundary of biophysics and nanomedicine: the observation of the internalization of different kinds of nanoparticles (boron-nitride nanotubes, barium-titanate nanoparticles and barium-titanate-core/gold-shell nanoparticles) by cultured cells and the demonstration of the nanopatterned nature of a structure built with two-photon lithography.