Quick and specific non-linear microscopy using shaped pulses with durations down to 10fs

Abstract

Nonlinear optical microscopy is ideally suited for in vivo imaging: it provides label-free contrast revealing intrinsic structural and chemical properties of the sample in a non-invasive way. Successful nonlinear microscopy relies on the use of pulsed lasers to obtain high signal levels at moderate average laser power. In particular, broadband excitation increases the nonlinear generation efficiency as well as the spectral coverage. However, lower photodamage thresholds and less straightforward signal interpretation have prevented its application to sensitive samples. In this thesis, a pulse shaper is used to tailor ultrashort pulses for optimal imaging. This work concentrates on Coherent Anti-Stokes Raman Scattering (CARS) because it provides an access to highly specific vibrational spectra. The main concept is to encode molecule-specific information directly in the excitation. This is realized either by direct tailoring in a shaperassisted variant of a Multiplex CARS setup or by phase shaping of a single ultrashort pulse (<10fs). The photon load reduction and the optimization of the pulse profile achieved by shaping are demonstrated with the imaging of polymer samples and sensitive biological tissue. The flexibility of the setup allows switching between spectrally resolved acquisition for precise chemical mapping and single channel detection for rapid imaging. Further nonlinear effects can likewise be controlled by pulse shaping. In this work, the systematic modification of the relative intensities of Second Harmonic Generation (SHG), Two-Photon Excited Fluorescence (TPEF) and CARS is investigated as well as selective excitation of fluorophores and molecular vibrations. Multimodal imaging with shaped ultrashort pulses proves to be particularly efficient for biological samples as illustrated by the imaging of plant cells and skin biopsies.