| Abstract |
Two Photon Fluorescence (TPEF), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG) are relatively new imaging tools for the imaging of biological structures and processes at the microscopic level. The combination of TPEF, SHG, and THG imaging contrast modes in a single instrument provides unique and complementary information concerning the structure and the function of tissues and individual cells. In the present study, a useful, reliable, compact set-up for the in vivo simultaneous TPEF, SHG and THG imaging of biological samples, was developed.
By means of the developed setup, high-resolution (~200nm) mapping of the nematode C. elegans in both its anterior and posterior body part was achieved. Unique information related the structure and the functions of tissues and cells of the nematode were extracted. Numerous strains were investigated. Wild type, mutants expressing GFP (membrane localized or cytoplasmic) in mechanoreceptor neurons, mutants expressing diffuse GFP in the pharyngeal cytoplasm and expressing diffuse DSRED in the head and motor neurons. Optimum conditions for the detection of THG signal, arising from the worm, were found. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. This is the first time that a THG modality is employed to image live C. elegans specimens.
Additionally, by performing THG measurements, neurodegeneration phenomena were investigated. The in vivo precise identification of the shape and the contour of the degenerative neurons in the posterior part of the nematode and the monitor, in real time, of the evolution of the degenerative neurons of the worm through THG imaging measurements were achieved.
The development and optimization of a user friendly and compact prototype microscope system that combines three different nonlinear image-contrast modes (TPEF-SHG-THG) in a single instrument will be useful for a variety of applications in the field of biology and medicine. It offers new insights into the studies of embryonic morphological changes and complex developmental processes. The excellent 3D resolution capacity of these non linear techniques allows us to capture the subtle developmental information on the cellular or subcellular levels occurring deep inside the live larvae. Moreover, THG imaging measurements provide a quantitative description of the dynamics of internal structures in unstained wild type and mutant embryos and model organisms.
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In vivo imaging of structural features and processes of C. elegans using non-linear microscopy (TPEF, SHG, THG)
