Multimodal-nonlinear-optical-microspectroscopy for biological and diagnostic applications

Abstract

Multimodal non-linear imaging techniques provide non-invasive and potentially in vivo means to investigate tissue with cellular resolution. A particularly promising approach that has garnered attention as of late is the combination of coherent antiStokes Raman scattering (CARS), second harmonic generation (SHG) and two photon excited autofluorescence (TPEF) microscopy. In the first section of this thesis, the diagnostic potential of multimodal non-linear imaging has been demonstrated in the case of head and neck squamous cell carcinoma. The second part of this thesis investigates the feasibility of CARS microscopy for imaging intense bands in the finger-print region (800-1800 cm 1) wherein the presence of multiple overlapping peaks and interference with non-resonant background present challenges. Specifically, the emphasis is on imaging the prominent peaks arising from conjugated C=C double bonds in retinol, tretinoin, β-carotene, and various microalgal pigments. The first CARS fingerprint imaging application in the thesis is concerned with the vitamin A content of liver tissue. Analogously, in a uni-cellular application, CARS has been employed to image carotenoids in the diatoms D. brightwellii and S. turris. As part of the effort in transferring multimodal microscopic technologies to the enduser, the third part of the thesis examines two beam excitation and demultiplexed detection as a means of doubling the speed of laser scanning microscopes based on compact fiber laser sources. Another area of improvement explored is the resolution of the CARS microscopic setup wherein, based on results from numerical studies, a Bessel like beam was employed as one of the excitation arms in the setup to enhance lateral resolution.