High Throughput High Sensitivity Depth Resolved Wide Field Microscopy

Abstract

3D optical microscopies including confocal microscopy, two-photon excitation microscopy, and coherent anti-Stokes Raman scattering microscopy have optical sectioning capability, but their image acquisition is relatively slow due to the sequential nature of raster scanning. Recently, scanningless nonlinear microscopy based on temporal focusing was introduced as an alternative to using the diffraction-limited spot. However, comparable optical sectioning has not been proved without optimizing the optical design and high-throughput capability has not been achieved due to the optical power limitation. In this presentation, high-throughput high-sensitivity depth-resolved wide-field two-photon microscopy is proposed. To quantify depth discrimination capability, a comprehensive mathematical model for depth-resolved wide-field illumination is derived and experimentally validated. By optimizing optical design parameters through numerical simulation, the best 3D resolution is shown to be close to diffraction limit. In addition, single particle detection sensitivity and high-throughput imaging capability are demonstrated by incorporating quantum dots, which are known to have high two-photon cross section, as a contrast agent into the proposed system. Finally, depth-resolved single particle tracking is evaluated to study the transport process in the cells with the developed microscopy, which confirms that this microscopy holds the potential in the fields of biology and medicine where both sensitivity and throughput are required.