Airy beam based two-photon microscopy

Abstract

Two-photon microscopy holds an important role in modern biological research. Based on two-photon fluorescent excitation, where the fluorescent molecule simultaneously absorbs two excitation photons and subsequently emits one fluorescent photon, high resolution and high contrast can be achieved in this imaging technique, with less optical damage and deep penetration due to the longer wavelength employed. Nowadays, two-photon microscopy has been widely applied to biological observations and biomedical studies. However, one limitation of conventional two-photon microscopy lies in the ability of imaging volumetric samples. The axial imaging range of two-photon microscopy is limited within a narrow band near the focal plane. In order to image a three-dimensional sample in conventional two-photon microscopy, a stack of image at different depth is needed, which generally requires an additional axial scan. This extra axial scan severely decreases the image acquisition speed, making it difficult to achieve fast volumetric imaging. In the thesis, diffraction-free Airy beam is employed to improve the volumetric imaging speed of conventional two-photon microscopy. As a result of diffraction-free nature, Airy beam produces a much longer focus along the axial direction in confocal system. Taking the advantage of the elongated Airy focus, the axial signals are projected to a single pixel. Therefore, a two-dimensional frame under Airy beam illumination actually records the projection of a stack of image at different axial position, which significantly increases the acquisition speed of volumetric specimen. Moreover, the penetration depth of two-photon microscopy is further increased by employing non-diffracting Airy illumination. Experimentally, a compact experimental setup is built to perform two-photon microscopy, which can be switched between Gaussian and Airy illumination mode. Basic imaging characteristics of the system are studied via measuring the point-spread functions, which show the Airy beam significantly extends the axial imaging range without scarifying the lateral resolution. Volumetric imaging is performed using fluorescent beads as well as real biological tissues, where a large depth of view is identified in Airy mode. A highly-scattering sample is fabricated to demonstrate the penetration ability, where Airy beam penetrates deeply into the sample due to its robustness against disturbance. To conclude, this work improves the deep volumetric imaging ability of conventional two-photon microscopy, which promotes the application of Airy beam and benefits biological studies.