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Non linear imaging at microscopic level for biological applications

Final Activity Report Summary - NOLIMBA (Non Linear Imaging at Microscopic level for Biological Applications)

Two advanced experimental workstations for the in vivo non-linear imaging microscopy of biological samples and for multiphoton polymerisation of biomaterials have been developed at IESL-FORTH in the framework of NOLIMBA project. For the imaging applications, within the framework of this project, the development and the optimisation of a user friendly, compact prototype microscope system, that combines different nonlinear image-contrast modes (Two Photon Excitation Fluorescence (TPEF), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG)) in a single instrument for biological applications, was achieved. 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. By means of the developed setup, high-resolution mapping of the nematode Caenorhabditis elegans (C. elegans) in both its anterior and posterior body part was achieved. Unique information related the structure and the functions of tissues (pharynx) and specific cell types (neurons) of the nematode were extracted. Numerous strains were investigated. Two and Three Dimensional reconstructions of nonlinear images from the biological samples were performed. Moreover, the in vivo, precise identification of the contour of the degenerating neurons in the posterior part of the nematode and the monitoring, in real time, of the progression of degeneration in the worm, through THG imaging measurements, were achieved.

Furthermore, during the last year of the project the non-destructive imaging technique of THG was employed for the characterisation of the different developmental stages of the nematode C. elegans embryos. THG images from different embryonic stages of C. elegans development (early embryonic stage, bean stage, 3-fold stage) were collected. Additionally, during the fourth year of the project SHG and THG polarisation dependence measurements to wild type C. elegans samples have been performed. THG signals proved to have no dependence on incident light polarisation, while SHG images are highly sensitive to the changes of the incident linearly polarised light. During the last year of the project, by employing multi-photon excitation fluorescence (MPEF) modality, the sub-cellular localisation of gold nanoparticles and new generation photosensitisers for Photodynamic Therapy (PDT) was accomplished.

There was an effort during the last year of NOLIMBA project for imaging optimisation of biological samples using non-linear excitation and pulse shaping techniques. Pulse shaping techniques correlated with a feed-back control algorithm were employed in order to increase the signal of SHG, and thus, to enhance the resolution of 3-D imaging of biological samples. This involved the combination of intense, ultrashort (25 fs) laser pulses at 800nm with a spatial light modulator (SLM) within a 4f optical imager to perform the pulse shaping in the temporal domain. Starch has been used as a model system.

The efficiency of using shaped femtosecond laser pulses in Second Harmonic Generation and signal increase has been proved, by performing these series of measurements. This technique could be implemented into the next generation devices for the investigation of living biological specimens. These initial results are very encouraging for continuation of experiments to more complicated and novel biological substances. In addition, the feasibility to use pulse shaping techniques to thin collagen films in order to obtain better control of ablation procedure was tested.

Regarding biosensor applications, during the duration of this project, a new method was developed for the precise, three-dimensional patterning of biomolecules. The technique involved the selective attachment of photosensitive biotin on 3D structures made by two-photon polymerisation and the subsequent immobilisation of biomolecules such as avidin and amyloid peptides. This method can be used for the development of three dimensional biosensors, and functionalised implants.