Nonlinear optical procedures for the diagnostics and processing of biological samples by using ultra-short laser pulses
Authors: Τσερεβελάκης Γεώργιος 
Published: August 2021

The nonlinear imaging techniques represent the forefront of research in cell biology. These modalities constitute a powerful tool for elucidating structural and anatomical changes of biological samples and for probing functions and developmental processes in vivo at the microscopic level. The investigation of in vivo cellular and sub-cellular activities, by means of these nonlinear imaging techniques, can provide novel information related to fundamental biological problems, leading to the development of innovative methodologies that can be useful for a variety of applications in the field of biology and medicine.Within the framework of this thesis, the development and the optimization of a user-friendly compact prototype microscope system that combines different nonlinear contrast modes such as Multiphoton Excitation Fluorescence and Optical Harmonics Generation (an analytical overview of which is given in the first two chapters) with the capability of performing nanosurgery experiments was achieved. The developed set-up was employed for various biological applications, extracting novel results.We initially demonstrated the great potential of label-free Third Harmonic Generation (THG) imaging microscopy for the characterization of different developmental stages in C. Elegans embryogenesis. Furthermore, cell tracking studies were performed in live, unstained embryos through the prolonged time-dynamic monitoring (up to 7 hours) of the mitotic cell divisions during early embryoegenesis. Thus, THG contrast modality was proven to be a powerful diagnostic tool, providing valuable information and offering new insights into the complex developmental process of C. Elegans embryogenesis.The encouraging results of the previous study were exploited further in the next section of the current work, where the following of the course of pre-implantation embryo patterning by nonlinear microscopy was successfully accomplished. More specifically, THG imaging, by detecting mitochondrial / lipid body structures, could give reliable information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.It is well-known that lipids are the main components of cell membranes, function as signalling molecules and are the main energy store of organisms. Excess energy is stored as fat in adipocytes leading to obesity. The energy control and metabolism pathways that control lipid metabolism are still unrevealed. For this reason, we developed an alternative to the common dye-based approaches methodology connected with nonlinear THG imaging, to visualize fat deposition using C. Elegans as a model organism. As it has already been mentioned, this approach is non-destructive and alleviates the requirement of staining the sample. We excluded the possibility that lipofuscin contributes to the THG signal and instead found that fat is the main contributor of high THG signal in the intestine of C. Elegans. To validate our approach, it was shown that multiphoton excitation fluorescence, following lipid staining with BodiPy 500/510, Nile Red and Oil Red-O and THG signals were colocalized in wild type worms. To further support the efficiency of THG in detecting lipid droplets, we showed that mutant worms deficient in FAT-7 and GLO-1 genes had fewer lipid droplets, while in DAF-2 had more lipid droplets compared to wild type animals. Finally, our study indicated that fat accumulated progressively until early adulthood, while it progressively decreased during the later stages of the worm lifespan. Consequently, within the framework of this study, THG imaging technique was proven as a potential innovative tool for the monitoring of important biological procedures related to the process of aging.In the last section of the present thesis, we utilized THG microscopy as a powerful diagnostic tool for the identification of structures that were subjected to nanosurgery experiments. Femtosecond laser assisted nanosurgery of microscopic biological specimens is a relative new technique which allows the selective disruption of sub-cellular structures without causing any undesirable damage to the surrounding regions. The targeted structures are usually stained with some specific dye in order to be clearly visualized for the nanosurgery procedure. However, the validation of the final nanosurgery result is quite difficult, since the targeted structures could be simply photobleached rather than selectively destroyed. This fact constitutes a main drawback of the fluorescence technique. On the other hand, in the case of THG imaging, no staining of the biological sample is required since THG is an intrinsicproperty of matter. By employing a multimodal system which integrates nonlinear imaging modalities with nanosurgery capabilities, the selective disruption of sub-cellular structures (most probably lipid droplets) in HeLa cancer cells was successfully achieved, proving thus the reliability of the THG technique. During the last part of the study, cells’ viability post nanosurgery procedure was verified via Two Photon Excitation Fluorescence (MPEF) measurements.In conclusion, nonlinear microscopy techniques have been proven to present a great potential not only in the fundamental biomedical research, but also in ‘real-world’ problems. Furthermore, this applicability has already been extended in the diagnosis and treatment of serious diseases, such as neurodegeneration, arterial disorders and cancer.