Design, analysis and aberration correction of high-resolution beam-scanning stimulated emission depletion microscopy

Abstract

Confocal microscopy has been a versatile tool for investigating various specimens in bio-science, material science, semi-conductor industry, and etc. Recently, it is widening the application to the bio-medical fields along with the confocal endo-microscopy. However, due to the well-known resolution limit in light microscopy, some researchers inevitably find alternatives, such as atomic force microscopy or scanning electron microscopy, enduring the complicated preparation, time-consuming process, sample damage, and limited use in fluorescent applications. Thus researchers have proposed many ideas to overcome the resolution limit by far-field imaging, such as 4-pi confocal microscopy, two-photon microscopy, structured illumination microscopy, point spread function engineering, stimulated emission depletion microscopy (STED microscopy), confocal self-interference microscopy, and etc. Among them the STED microscopy is a promising technique for high spatial resolution. It achieves the higher resolution over the conventional confocal microscopy, by applying additional STED pulse only at the outer region of the excitation pulse. STED pulse depletes the excited fluorophore by the depletion process, named stimulated emission depletion, thus it leads the reduction of full-width at half-maximum of the effective intensity point spread function. It has theoretically unlimited resolution. The experimental verification of the resolution shows that 30nm in both transverse directions have been achieved and about 30nm in axial direction has been acquired, individually. One of the mainly required techniques in the STED microscopy is applying the beam-scanning optics. Until now the STED microscopy uses the sample scanning method to require 2- or 3-dimensional image. This causes the slow scan speed and vibrational problems. Thus, in this study the beam-scanning STED microscopy is proposed. In order to scan the laser beam, the two-axis galvanometers are used since it has suffi...