3D reconstruction of high‐resolution STED microscope images

Abstract

Tackling biological problems often involves the imaging and localization of cellular structures on the nanometer scale. Although optical super-resolution below 100 nm can be readily attained with stimulated emission depletion (STED) and photoswitching microscopy methods, attaining an axial resolution <100 nm with focused light generally required the use of two lenses in a 4Pi configuration or exceptionally bright photochromic fluorophores. Here, we describe a simple technical solution for 3D nanoscopy of fixed samples: biological specimens are fluorescently labeled, embedded in a polymer resin, cut into thin sections, and then imaged via STED microscopy with nanoscale resolution. This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes.

Signalling complexes and clusters: functional advantages and methodological hurdles

“…Similar progress has also been reported for STED microscopy Punge et al, 2008). Despite the severe time constraints of current nanoscopy techniques, some attempts have been made to study dynamic events in live cells at video-rate acquisition speed (Conley et al, 2008;Manley et al, 2008;Westphal et al, 2008).…”

Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

“…Previous subdiffraction optical studies in neurons (31,36) and rat adrenal gland phaeochromocytoma (PC12) cells (37, 38), using stimulated emission depletion (STED) imaging in both fixed and live specimens, have also indicated the presence of nanometer-sized clusters for a variety of membrane or membrane-associated proteins, including syntaxin-1 and SNAP-25. Notably, although sequestration of such proteins seems to be a general paradigm, the resolution (30-to 80-nm FWHM) of those experiments was inadequate to directly resolve multiple copies of a protein in each nanodomain or to directly reveal details of the architecture of such domains.…”

“…Additionally, several multicolor proof-of-concept experiments have been described in systems with well-defined spatial organization (28)(29)(30)(31)(32)(33), in which interpreting images of a priori known structures has been straightforward. However, due to experimental inaccuracies in registering multicolor single-molecule localizations and the lack of an analytical framework to extract biologically meaningful information from such data, colocalizing two irregularly distributed protein species on length scales below ∼100 nm, down to the length scales associated with formation of distinct molecular complexes, has proved technically challenging.…”

Super-resolution microscopy at a glance

“…Although the simplest way to generate 3D super-resolution images is to combine the serial sectioning of tissue with standard lateral super-resolution techniques (Punge et al, 2008), many superresolution techniques have now been extended to 3D. In 3D STORM a cylindrical lens is simply added in the light path to create astigmatic imaging so that the ellipticity of the PSF becomes a sensitive measure of its distance from the focal plane, and yields a resolution of 20-30 nm laterally and 50-60 nm axially (Huang et al, 2008).…”