Resumen de Applications of two-photon fuorescence in biology and immunosensing
Anisha Thayil Karunakaran Nair
In recent years, non-linear optical methods have been successfully applied for the imaging of biological specimens and this development has revolutionized many areas of life science research. The primary goal of the present study is the design and construction of a laser scanning two-photon excited fluorescence (TPEF) microscope in the laboratory. Its custom design allows flexibility to use microscope in an active way. One of its key features is the integration of different nonlinear imaging modalities like TPEF, second and/or third harmonic generation and the possibility to induce controlled micro-ablations in biological specimen. This thesis then discusses an important aspect of multiphoton microscopy, which is the characterization of the spectral and temporal features of the laser ?eld at the sample plane of a high NA, TPEF microscope. A detailed knowledge of the ultra-short pulses that interact with the biological specimen is important to optimize imaging and to reduce photodamage. In this respect, we have implemented a simple methodology that makes use of the backward propagating second- harmonic generation (SHG) signal from starch granules. Without any modification to the microscope, SHG-autocorrelation traces are obtained by using a single starch granule that was placed alongside the biological specimen being imaged. A spectrally resolved SHG autocorrelation has been acquired by placing a spectrometer at the output port of the microscope. Complete in-situ pulse information is then directly retrieved in an analytical way using the measurement of electric filed by interferometric spectral trace observation (MEFISTO) technique. We have shown that adapting a standard single mode fiber and a pair of prisms to the microscope, the pulses arriving at the sample plane can be compressed by a factor of 5, thereby improving the two-photon excitation efficiency. On the application side, we use the TPEF microscope for combined imaging and laser tissue ablation of a living Drosophila Melanogaster embryo. By using tightly focused near infrared femtosecond pulses at MHz repetition rate and of subnanojoule energy we are able to perform microsurgery on the epithelial tissue with in Drosophila embryo at the final stages of its embryonic development. Tissue ablation has been performed on labeled and unlabeled embryos, during and after the process of dorsal closure. Our observations show that the ablation of GFP labeled tissue requires lower energy deposition than unlabeled tissue, ensuring that the tissue ablation is mediated by multiphoton absorption of GFP. In addition, the energy deposition to produce ablation is lower during dorsal closure. This results show the presence of additional tensile forces on the tissue during dorsal closure. We have also observed that Drosophila epidermis try to heal from the laser wounds with increased activity of actin near the wound edges. An important implication of these experiments is to understand the genetic signaling mechanism during epithelial wound healing. A few attempts have been made in this direction to check the activation of puckered gene, a target of the C-Jun NH2- terminal kinase (JNK) signaling pathway. Our preliminary observations suggest that puckered is activated on a single row of cells around the epithelial laser wound, indicating the activation of JNK pathway. Apart from the microscopy applications, it was the aim of this thesis to use TPEF spectroscopy as an immunosensing scheme. To achieve this goal, we have used grating waveguide structures as an enhancement platform for two-photon excitation. The evanescent ?eld ampli?cation property of this resonant device allows enhanced TPEF excitation over larger area without the need for tight focusing. Using such dielectric, resonant double grating waveguide structures (DGWS), we have observed a 330- fold enhancement of the integrated TPEF signal in TMR thin ?lms compared to the conventional focussing methods. This together with the unique advantages of TPEF spectroscopy allows signal detection from down to100 picomolar solutions. We have further demonstrated, for what we believe is the first time, the use of TPEF spectroscopy as a detection method of a? nity binding reactions. Using resonant DGWS we have developed a direct competitive assay scheme for detecting methyl boldenone, a synthetic anabolic steroid. A limit of detection down to 0.1ppb has been reached, which is well below of the minimum required performance limit (MRPL) required from World Anti -Doping Agency. In conclusion, we have shown that TPEF microscopy, being a relatively non-invasive high resolution 3D imaging tool has immense potential in areas such as developmental biology.
Furthermore, we have demonstrated that by exploiting the advantages of TPEF combined with resonant structures, a TPEF immunosesnor can become an important tool in doping and food safety controls.
