3 Color - 3 Dimensional STED Nanoscopy

The stimulated emission depletion microscopy (STED) is a super-resolution technique that enables to overcome the diffraction barrier, allowing to distinguish details of cellular as well as molecular structures, not visible with a conventional confocal microscope [1]. Here we present the development of a custom made versatile STED microscope, based on a super-continuum pulsed laser source endowed with 2 high power STED laser beams, at 715 and 745 nm, respectively [2]. In order to obtain three-dimensional super-resolved images, we split the STED beam into two parts. One of the beams passes through a vortex phase plate which creates a donut shape beam for lateral resolution enhancement. For the other beam we introduced a home-made phase plate which introduces a phase delay of π in the center, creating a z-donut to increase resolution in the axial direction [3]. We can tune the microscope PSF adjusting the ratio of the power in the two depletion pathways, to achieve, for example, an isotropic resolution. Additionally in order to perform three color imaging we paid particular attention to chromatic aberrations and we optimized the setup accordingly. Therefore by using proper fluorophores, we are able to do 3 color super-resolved imaging. Thanks to this completely custom made optical architecture, designed for fast scanning, 3D and three color acquisition, we are able to perform multimodal live cell imaging. The achieved 3D resolution discloses morphometric properties at the nano-scale which are completely hidden to the confocal observation.