Abstract
Stimulated emission depletion (STED) nanoscopy is one of a suite of modern optical microscopy techniques capable of bypassing the conventional diffraction limit in fluorescent imaging. STED makes use of a spiral phase mask to enable 2D super-resolution imaging whereas to achieve full volumetric 3D super-resolution an additional bottle-beam phase mask must be applied. The resolution achieved in biological samples 10 µm or thicker is limited by aberrations induced mainly by scattering due to refractive index heterogeneity in the sample. These aberrations impact the fidelity of both types of phase mask, and have limited the application of STED to thicker biological systems. Here we apply an automated adaptive optics solution to correct the performance of both STED masks, enhancing robustness and expanding the capabilities of this nanoscopic technique. Corroboration in terms of successful high-quality imaging of the full volume of a 15µm mitotic spindle with resolution of 50nm x 50nm x 150nm achieved in all three dimensions is presented.
Highlights
Fluorescent nanoscopy has opened up a range of new scientific directions in the biological sciences as well as in optical physics and engineering
The samples were imaged in the adaptive optics (AO) 3D Stimulated emission depletion (STED) microscope, which has three imaging modes: confocal, 2D STED and 3D STED
Mitotic spindle images were acquired for three different microscope modes: confocal, 2D STED and 3D STED (π-step phase mask)
Summary
Fluorescent nanoscopy has opened up a range of new scientific directions in the biological sciences as well as in optical physics and engineering. Stimulated emission depletion (STED) microscopy [1] is one such technique, and has been shown to successfully resolve structures as small as 20nm in biological samples [2] whereas 5nm resolution has been demonstrated for imaging nitrogen vacancy colour centres in nano-diamonds [3,4]. For generating appropriate beam shapes, a specially designed mask is applied to the depletion beam in order to change its phase. For 2D imaging a doughnut beam such as a Laguerre-Gaussian beam (vortex beam) is used [7], while introducing a π-step phase change produces the bottle-beam [8] that will deplete out-of-focus light, increasing the axial resolution [9] and giving 3D capability. Axial resolution enhancement can be obtained with the combination of STED microscopy with 4-Pi microscopy [11,12,13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
