Second-harmonic-generation Microscopy Excited by Polariza- Tion Distribution Controlled Beam

We have developed a second-harmonic-generation (SHG) microscopy system whose electric filed at the focus is controlled three-dimensionally in order to observe three-dimensional molecular orientation with high spatial resolution. Figure 1 shows the developed system. The system consists of a femtosecond laser to induced nonlinear optical phenomenon (SHG), a parallel-aligned liquid-crystal spatial-light-modulator (PAL-SLM) to control polarization distribution on the cross-section of the excitation laser beam, and a transmission type scanning microscope to observe SHG light.[1] The femtosecond laser beam is reflected two times by the PAL-SLM. The first reflection controls the phase distribution of the beam, and the second reflection controls polarization angle distribution. As the result, we can convert the incident linearly polarized beam to orthogonal linearly polarized beam and the radially polarized beam without mechanical moving parts[2]. Since the linearly polarized beam generates perpendicular electric filed to the optical axis at the focus and radially polarized beam generates parallel electric filed, we can control the electric field at the focus three-dimensionally. SHG is forbidden in the material with inversion symmetry and is sensitive to the molecular orientation. Besides, SHG gives threedimensional spatial resolution by its nonlinearly. Hence, we can obtain the vector filed like image of the molecular orientation. We observed two samples of sliced Achilles' tendon, which mainly consists of collagen fibers. The directions of the collagen fibers are perpendicular (Fig. 3 (a)~(c)) and parallel (Fig. 3 (d)~(f)) to the sliced surfaces. It is found that the directions of the collagen fibers are determined by the excitation beam polarization. [1] K. Yoshiki, M. Hashimoto, and T. Araki, Jpn. J. Appl. Phys, 44, L1066-1068 (2005). [2] M. Hashimoto, K. Yamada, and T. Araki, Opt. Rev., 12, 37-41 (2005). Fig. 1 Developed microscopy system