TWO-PHOTON FLUORESCENCE AND SECOND HARMONIC GENERATION OF BIOTIN AND BIOTIN ESTER MICROCRYSTALS IN OPTICAL TWEEZERS USING AN IR PULSED LASER

Abstract – Intense second harmonic generation (SHG) was observed from microcrystals of biotin and biotin ester trapped in optical tweezers with an infrared pulsed laser beam serving for both trapping and photo-excitation. The use of nonlinear optical signals in laser scanning microscopy has become a subject of tremendous interest. Many methods and advancements in optical microscopy were developed based on nonlinear optical signals. There are basically two types of nonlinear optical signals, one is incoherent, such as multiphoton fluorescence, and the other is coherent, such as second or third harmonic generation. In the former case, fluorescence photons are emitted from molecules excited to a higher energy level by simultaneous absorption of two or more photons. The resulted emission spectrum is usually similar to what is observed in single photon excitation, in which the emission usually depends only on the molecule’s inherent properties and is largely independent of the energy of the excitation photon. In the case of two-photon (2-p) fluorescence excitation, the intensity varies linearly with the concentration of the fluorophores and is quadratic with the peak incident power [1]. In harmonic generation, the irradiated molecule does not experience a real transition. Second harmonic generation is a second-order nonlinear optical process that arises from the medium’s inherent asymmetrical response to an applied electric field, e.g. within a nonlinear (non-centro-symmetric) crystal or at an interface that possesses broken symmetry. A coherent wave is generated at exactly twice the incident frequency when an intense lase