The requirement for imaging living structures with higher contrast and resolution has been covered by the inherent advantages offered by nonlinear microscopy (NLM). However, to achieve its full potential there are still several issues that must be addressed. To do so, it is very important to identify and adapt the key elements in a NLM for achieving an optimized interaction among them. These are 1) the laser source 2) the optics and 3) the sample properties for contrast generation. In this thesis, three strategies have been developed for pushing NLM towards its limits based on the light sample interaction optimization.

In the first strategy it is experimentally demonstrated how to take advantage of the sample optical properties to generate label-free contrast, eliminating the requirement of modifying the sample either chemically or genetically. This is carried out by implementing third harmonic generation (THG) microscopy. Here, it is shown how the selection of the ultrashort pulsed laser (USPL) operating wavelength (1550 nm) is crucial for generating a signal that matches the peak sensitivity of most commercial detectors. This enables reducing up to seven times the light dose applied to a sample while generating an efficient signal without the requirement of amplification schemes and specialized optics (such as the need of ultraviolet grade). To show the applicability of the technique, a full developmental study of in vivo Caenorhabditis elegans embryos is presented together with the observation of wavelength induced effects. The obtained results demonstrate the potential of the technique at the employed particular wavelength to be used to follow morphogenesis processes in vivo.

In the second strategy the limits of NLM are pushed by using a compact, affordable and maintenance free USPL sourcet. Such device was designed especially for two-photon excited fluorescence (TPEF) imaging of one the most widely used fluorescent markers in bioimaging research: the green fluorescent protein. The system operating parameters and its emission wavelength enables to demonstrate how matching the employed fluorescent marker two-photon action cross-section is crucial for efficient TPEF signal production at very low powers. This enables relaxing the peak power conditions (40 W) to excite the sample. The enhanced versatility of this strategy is demonstrated by imaging both fixed and in vivo samples containing different dyes. More over the use of this laser is employed to produce second harmonic generation images of different samples. Several applications that can benefit by using such device are outlined. Then a comparison of the employed USPL source is performed versus the Titanium sapphire laser (the most used excitation source in research laboratories). The final goal of this strategy is to continue introducing novel laser devices for future portable NLM applications. In this case, the use of chip-sized semiconductor USPL sources for TPEF imaging is demonstrated. This will allow taking NLM technology towards the sample and make it available for any user.

In the last strategy, the light interaction with the optical elements of a NLM workstation and the sample were optimized. The first enhancement was carried out in the laser-microscope optical path using an adaptive element to spatially shape the properties of the incoming beam wavefront. For an efficient light-sample interaction, aberrations caused by the index mismatch between the objective, immersion fluid, cover-glass and the sample were measured. To do so the nonlinear guide-star concept, developed in this thesis, was employed for such task. The correction of optical aberrations in all the NLM workstation enable in some cases to have an improvement of more than one order of magnitude in the total collected signal intensity.

The obtained results demonstrate how adapting the interaction among the key elements of a NLM workstation enables pushing it towards its performance limits.


Wednesday March 27, 11:00. ICFO Auditorium

Thesis Advisor: Prof. Pablo Loza Alvarez