Optical microscopy uniquely provides non-invasive imaging of biological specimens and has become an essential tool in life sciences. So far, fluorescence microscopy techniques have been the most widely used due to their ability to visualize the molecules of interest with high contrast, high specificity, and high spatial and temporal resolution. In the last decades, much effort was put into the development of advanced super-resolution fluorescence microscopy techniques to circumvent the diffraction limit and image with unprecedented spatial resolution. The non-linearities contained in saturation phenomena were exploited for this purpose, and led to the development of techniques like stimulated emission depletion (STED) and saturated excitation (SAX) microscopy, which proved to have the potentiality to really break the diffraction barrier and tune the resolution down to infinitely small focal volumes (Chapter 1). Despite their well-established benefits, (super-resolution) fluorescence microscopy techniques rely on the photophysical properties of fluorescent molecules to obtain the desired contrast and resolution, and the labeling procedures may alter the physical properties of the specimen and may come at the cost of photobleaching and photodamage effects. With the development of ultrashort pulsed laser sources, new types of non-linear optical interactions became accessible (Chapter 1) and started to acquire a central role in optical microscopy for label-free imaging, providing novel non-fluorescence-based contrast mechanisms which purely rely on intrinsic properties of the molecules of interest. In this way, the invasiveness and the phototoxicity can be reduced, and the degradation of the fluorescence signal due to photobleaching can be avoided. Moreover, non-linear optical microscopy techniques allow for three-dimensional imaging due to the intrinsic optical sectioning capabilities of non-linear phenomena, while the use of longer wavelengths in the near-infrared part of the spectrum results in lower absorption and scattering and permits to image deeper inside tissues (Chapter 1). The main drawback of these non-linear optical microscopy techniques is their relatively poor spatial resolution, especially when using longer excitation wavelengths, and the fact that the imaging of non-fluorescent species with sub-diffraction resolution is still a challenging task. In this framework, this work aims at extending the super-resolution approaches to label-free microscopy techniques, based on the fact that, in principle, any saturable optical process between molecular states, not necessarily involving fluorescent transitions, is a potential candidate for breaking the diffraction barrier. In particular, non-linear optical processes are here exploited in near-infrared pump-probe microscopy techniques, such as transient absorption microscopy (TAM) and stimulated Raman scattering (SRS) microscopy (Chapter 3). In these techniques, two synchronized femto/picosecond pulsed laser beams are used to investigate ultrafast electronic and vibrational dynamical properties of the sample with high spatial and temporal resolution, high sensitivity and high chemical specificity. The interaction with the sample is recorded as an intensity variation of one of the two beams, which is extracted from the background adding a fast intensity modulation and filtering with a lock-in amplifier. New dynamical and chemical information can be accessed at the molecular level and in a label-free way, using intrinsic biomolecules as natural contrast agents. Absorption-based pump-probe microscopy is here optimized to retrieve structural and dynamical information from graphene-based samples with high sensitivity, and its conventional configuration is combined with an additional doughnut-shaped pump beam, which allows for the reduction of the effective focal volume exploiting transient absorption saturation. By optimizing the experimental parameters, such as power and temporal overlap of the saturation beam, single layer graphene deposited on a glass surface can be imaged at the nanoscale (Chapter 4). Moreover, the saturation of the vibrational excitation in SRS microscopy is here theoretically and experimentally assessed, in order to evaluate the applicability of the SAX approach to achieve isotropic sub-diffraction imaging capabilities (Chapter 5).
|Titolo della tesi:||Exploiting optical non-linearities for super-resolution label-free optical microscopy|
|Data di discussione:||26-mar-2019|
|Appare nelle tipologie:||Tesi di dottorato|