As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

<p>Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.</p> <p>In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.</p> / Dissertation

Nonlinear optical microscopy (NLOM) offers many advantages when imaging intact biological samples. By using ultrafast lasers in the near infrared and two photon excitation (TPE), signal production is limited to the focal volume and provides an excellent means for rendering thin, microscopic images from within the sample. Exogenous fluorophores/lumiphores may be used as efficient contrast agents to tag specific targets and provide enhanced signal. The efficiency of the TPE process in these contrast agents is broadly assumed to vary inversely with the laser pulsewidth, τ. In this work, we investigate the TPE efficiency of transform limited broadband (~133nm, ~10fs) and narrowband (~11nm, ~170fs) pulses in the generation of twophoton luminescence from semiconductor nanocrystals or quantum dots (QD’s) both theoretically and experimentally. Compared to standard organic dyes, QD’s possess a relatively broad, uniform spectral response that enables better use of the full bandwidth from the broadband laser. Theoretical calculations including both degenerate and non-degenerate TPE indicate a rolloff from the 1/τ behavior as the pulses’ spectral bandwidth becomes broader than the absorption spectra of the QD’s. Experimentally measured enhancement in luminescence intensity while using a broadband pulse is compared with the simulated enhancement in two-photon luminescence. A combination of increased understanding of the excitation processes in NLOM and proper selection of contrast agents will help in advancing the role of broadband ultrafast lasers in NLOM.

The technique of hyper-Rayleigh scattering is employed to measure the first molecular hyperpolarizabilities£]of nonlinear optical chromophores provided by Industrial Technology Research Institute. £]0. In the thesis, the effect of the molecular structure and the solvent on the hyperpolarizabilities will be discussed further.

Nonlinear optical microscopy (NLOM) offers many advantages when imaging intact biological samples. By using ultrafast lasers in the near infrared and two photon excitation (TPE), signal production is limited to the focal volume and provides an excellent means for rendering thin, microscopic images from within the sample. Exogenous fluorophores/lumiphores may be used as efficient contrast agents to tag specific targets and provide enhanced signal. The efficiency of the TPE process in these contrast agents is broadly assumed to vary inversely with the laser pulsewidth, τ. In this work, we investigate the TPE efficiency of transform limited broadband (~133nm, ~10fs) and narrowband (~11nm, ~170fs) pulses in the generation of twophoton luminescence from semiconductor nanocrystals or quantum dots (QD's) both theoretically and experimentally. Compared to standard organic dyes, QD's possess a relatively broad, uniform spectral response that enables better use of the full bandwidth from the broadband laser. Theoretical calculations including both degenerate and non-degenerate TPE indicate a rolloff from the 1/τ behavior as the pulses' spectral bandwidth becomes broader than the absorption spectra of the QD's. Experimentally measured enhancement in luminescence intensity while using a broadband pulse is compared with the simulated enhancement in two-photon luminescence. A combination of increased understanding of the excitation processes in NLOM and proper selection of contrast agents will help in advancing the role of broadband ultrafast lasers in NLOM.

We combined an ECDL and the fluorescence detection system into a portable frequecy-stabilized laser.We installed an atomic cesium cell inside rather than outside the external diode laser cavity in order to minimize the frequency-stablized laser to palm size.In this way, we improved the mobility of the frequecy-stabilized laser. Moreover, the enhanced intensity of laser inside the external cavity increased the signal to noise ratio of the error signal for feedback control. We used Allan variance as an efficient method to quantify noise of the laser and we found the best value was 2.9954E-12 when the time integration was 90 second and the line width of laser was 200 kHz by beat frequency. This design can be exploited to lock the offset frequency of a femtosecond frequency comb laser, thus being helpful in the reaserch of CEP(carrier-envelope phase) light source.

Μελέτη διφωτονικά διεγερμένου φθορισμού για τον υπολογισμό των ενεργών διατομών διφωτονικής απορρόφησης οργανικών μορίων. / Study of the two photon excitation fluorescence for the estimation of two photon cross sections of organic molecules

Στην παρούσα εργασία, γίνεται αρχικά η μελέτη τριών χρωστικών ως προς την απόδοσή τους στη διφωτονική απορρόφηση. Με βάση τα αποτελέσματα της μέτρησης αυτής, επιλέχθηκαν οι δύο χρωστικές που παρουσίασαν ισχυρότερα το φαινόμενο, ώστε να μελετηθούν σχετικά με τον αποχρωματισμό τους. Για το λόγο αυτόν, τα δείγματα που παρασκευάστηκαν από τις ουσίες, δέχτηκαν ακτινοβολία μεγάλης έντασης, μέσω πολύ στενών παλμών laser. / Photobleaching of organic dyes after two photon excitation

Two-photon absorption is the process in which a molecule absorbs two photons simultaneously. The two key advantages of two-photon processes over one-photon processes are the possibility of excitation of materials with high three-dimensional spatial resolution and deep light-penetration into absorbing materials. Based on bond-cleavage reactions activated by photon-induced intramolecular electron transfer, two-photon activatable acid and radical initiators and two-photon removable protecting groups have been successfully designed and synthesized for photopolymerization and three-dimensional microfabrication and for biomedical photo-triggers. The optical and chemical properties of synthesized molecules, such as quantum yield of acid generation, initiation efficiency of photopolymerization, and photolysis efficiency, have been studied by using a variety of physical and analytical techniques under one-photon conditions. The two-photon characteristics and applications of these molecules are being investigated in collaboration with other groups.

Nonlinear optics has had extensive application into a vast array of scientific fields. One such nonlinear process, two-photon absorption (TPA), has had a wildly successful adoption into the field of biological imaging and microscopy. As far and as fast as this field is progressing, limitations stemming from the use of ultrafast lasers are starting to appear. In this work, an alternative nonclassical light source will be motivated for the application of low photon-flux two-photon microscopy. The origin and properties of the chosen nonclassical source, spontaneous parametric downconversion (SPDC), will be discussed along with the spatial and spectral properties modelled. Nonlinear processes such as TPA and sum frequency generation (SFG) will be viewed as "ultrafast coincidence measurements" of two photons arriving at a molecule within the time window of excitation. These ultrafast coincidence measurements will be viewed in an alternative manner: in terms of the second-order coherence from a light source. This degree of second-order coherence can be subdivided into two categories arising from different combinations of correlations within and between entangled photon pairs. Of interest, the energy-time correlations within the photon pair allow for enhancements in ultrafast coincidence rates over coherent light sources. The makings of an experimental setup to demonstrate enhanced rates from ultrafast two-photon coincidences taking place in SFG in a nonlinear crystal will be discussed.