Non-Linear Optical Process for Label-Free Nanoscopy

Optical microscopy is one widely used tool to study morphological and functional characterization of the observed, typically fluorescent-labeled specimen. Since labels may perturb the function of biomolecules, a recent challenge in bioimaging is to provide label-free imaging of complex biological systems in real-time and non-invasive mode. Investigating the structure of complex biological systems typically demands high speed, sensitivity, spectral and multi-contrast capability, temporal and spatial resolution. The demand of multimodal label-free imaging calls for developing a microscope that can enable fast, multi-contrast label-free, and non-invasive imaging. Today, a non-linear optical microscope can be a solution because it offers a multimodal multicontrast approach, although the assemble and optimization of the optical setup remains a challenge. While the non-linearity inherent when saturation phenomena occur is exploited to also achieve super-resolution label-free imaging, the development of ultrashort and ultrafast pulsed laser sources has led non-linear optical (NLO) light-matter interaction become accessible (Chapter 1). It paved the way to reach label-free imaging relying on the intrinsic properties of specimens. Label-free is not invasive nor destructive, thus suitable for living cell and tissue imaging. The implementation of NIR illumination led to developing a multimodal microscope (Chapter 2) by combining the conception of non-linear processes (Chapter 1) to obtain multi-contrast imaging. Combined (multi-modality) instead of individual (single-modality) and multi-spectral (multi-color) imaging provide new and complementary structural and compositional information about the specimen. Among non-linear microscopy techniques, pump-probe microscopy can be considered a label-free and multicontrast imaging. In pump-probe microscopy, applying two pulsed laser beams with different frequencies enables exploring different non-linear processes through one platform, including sum-frequency generation, second-harmonic generation, multi-photon, transient absorption, and stimulated Raman scattering (Chapter 3). Despite the advantages of non-linear techniques, i.e., 3D sectioning capability and label-free acquisition, they present low spatial resolution, especially for longer excitation wavelengths. Currently, techniques like super-resolved transient absorption result in achieving a resolution enhancement (<100 nm), but further improvements remain challenging. In this thesis, I present a multimodal microscope that allows for simultaneous image acquisition from multiple optical imaging modalities, which we have constructed in Nanoscopy group at Center for Human Technology (CHT) of Istituto Italiano di Tecnologia (CHT), Genoa. The microscope enables the following methods: multi-photon excitation fluorescence (MPEF), sum-frequency generation (SFG), second harmonic generation (SHG), pump-probe (PP), and saturated pump-probe (SPP). The unique configuration of the integrated microscope allows for the acquisition of both scattered and transient absorption-based imaging information with particular emphasis in the fields of label-free super-resolution imaging through applying saturation absorption (Chapter 4). I focus on the optimization of such custom-built femtosecond-pulsed near-infrared pump-probe microscope for imaging biological and material specimens. Specifically, I used the following contrast mechanisms in a multimodal simultaneous approach: autofluorescence, SFG, SHG, and pump-probe. I applied the imaging methods at different biological targets, i.e., collagen, myosin, and cells. In addition, we modified the setup to collect also stimulated-Raman scattering from tendon, and I studied how the efficiency changes by varying the pairs of wavelengths (pump and probe), and Raman shifts. Furthermore, we explored the saturation of transient absorption process using a doughnut-shaped beam, in a STED-like approach, in the imaging of single-layer Graphene (SLG) and NP-Polycarbonate. The results showed that it is possible to obtain label-free super-resolution imaging of SLG and polycarbonate. I proposed a model based on Runge-Kutta fourth-order algorithm to estimate the required excitation power to reach absorption saturation by solving Liouville-von Neumann equations numerically. Finally, I numerically and experimentally presented how the pump-probe signals of SLG depend upon increasing illumination intensity varying the pump and probe intensities, while the other was kept constant. (Chapter 4). We demonstrated multiple techniques for multimodal label-free imaging of Huntington’s disease (HD) in the cellular model to achieve complementary structural and composition information about this disease. We performed white light quantitative 3D phase imaging of HD using a PRISM multi-plane custom optical microscope made in the nanoscale biology (LBEN) laboratory at EPFL. Next, we explored stimulated-Raman scattering (SRS) imaging at fixed positions in the samples through our custom-built near-infrared pump-probe microscope in the Nanoscopy group at IIT to study the chemical composition of HD. Finally, we applied FLIM techniques to measure autoflourescence lifetime of the samples (Chapter 5). In this framework, this work aims to improve structural content information about the Huntington aggregation in the cellular model under physiological-like conditions by applying label-free imaging approaches.