3D reconstruction of high-resolution STED microscope images.
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.
Punge
Annedore
A
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Rizzoli
Silvio O
SO
Jahn
Reinhard
R
Wildanger
Jan Dominik
JD
Meyer
Lars
L
Schönle
Andreas
A
Kastrup
Lars
L
Hell
Stefan W
SW
D000818:Animals; D022082:Cellular Structures / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D017785:Photons