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Two-Photon Microscopy: From the Optimisation of Fluorescent DNA Labels to Local Probe Scanning Second Harmonic Generation Microscopy
C. Fiorini-Debuisschert, I. Berline, G. Metgé, F. Charra, M. Mihaly, C. Allain, G. Bordeau and MP Teulade-Fichou

We have studied the two-photon absorption performances of specifically engineered triphenylamine derivatives bearing different acceptors (mono-, bis or trisubstitution). Large two-photon absorption cross sections (δ) could be evidenced. These compounds happen also to be weakly emissive in a buffer medium whereas a strong fluorescence signal is induced by addition of DNA, making them very interesting candidates for DNA labelling and high contrast two-photon induced fluorescence microscopy. As a complementary characterization tool for the study of biological systems, we propose an original technique which takes profit of Second Harmonic Generation (SHG) effects in molecular solutions. We show that we can use the electric field present inside a Scanning Tunneling Microscope (STM) junction towards creating a local noncentrosymmetry via molecular orientation under the tip. Experiments were performed inside a STM junction immersed in concentrated solutions of azo-dyes molecules chosen for their highly nonlinear properties and the possibility to generate a local SHG signal from those molecules was demonstrated. The potentialities of such technique towards high resolution imaging are discussed.

Keywords: Two-photon induced fluorescence, fluorescent label, electric field induced second harmonic generation, nonlinear microscopy.

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