Lifetime based axial contrast enable simple 3D-STED imaging

Abstract

Abstract Stimulated Emission Depletion (STED) microscopy increase spatial image resolution by laterally sharpening the illumination profile of the confocal microscope. However, it remains compromised in axial resolution. To improve axial STED resolution, constructive interference of the STED depletion beam must be formed surrounding the focal plane to turn off the fluorophores beyond the focal plane. For isotropic 3D-STED resolution, this axial STED interference pattern must be overlayed with the doughnut STED beam at nanometer accuracy. Such optical configurations can be challenging in alignment. In this current work, we introduced a straightforward lifetime based axial contrast in STED microscope by imaging the samples on an ITO coated glass coverslip. The STED laser generates surface plasmon resonance on the ITO surface that enhanced the metal induced energy transfer MIET effect on the ITO surface. The enhanced MIET effect established a lifetime gradient with ∼20% dynamic range that extend for mor than 400 nm from the ITO surface. The axial contrast based on the lifetime gradient was directly used for 3D-STED imaging of tubulin fibers inside COS-7 cells, where the vertical displacement of single tubulin fiber was revealed. Lifetime gating could be applied to further improve lateral spatial resolution. Considering that most common implementation of STED microscopes uses pulsed lasers and timing electronics, there is no optical modification of the microscope is required in the current 3D-STED approach.