Abstract
SummaryFluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.
Highlights
Major advances in cell biology are tightly linked to innovations in microscopy
single-molecule switching nanoscopy (SMSN) methods such as photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM) use a stochastic approach where only a small subset of fluorescent molecules is switched on at any particular moment in time while the majority remains in a non-fluorescent ‘‘dark’’ or ‘‘off’’ state (Gould et al, 2012a)
Super-resolved images are reconstructed from the positions of thousands to millions of single molecules that have been recorded in thousands of camera frames
Summary
Major advances in cell biology are tightly linked to innovations in microscopy. The development of fluorescence microscopy, for example, enabled sub-cellular localization of labeled proteins of interest (Lichtman and Conchello, 2005). The advent of super-resolution fluorescence microscopy, or nanoscopy, techniques such as stimulated emission depletion (STED) (Hell and Wichmann, 1994) and single-molecule switching nanoscopy (SMSN) (Betzig et al, 2006; Hess et al, 2006; Rust et al, 2006) has extended the application range of fluorescence microscopy beyond the diffraction limit, achieving up to 10-fold improvement in resolution (Gould et al, 2012a) These methods are maturing and offering the opportunity to observe biological phenomena never before seen (Chojnacki et al, 2012; Kanchanawong et al, 2010; Liu et al, 2011; Xu et al, 2013). Super-resolved images are reconstructed from the positions of thousands to millions of single molecules that have been recorded in thousands of camera frames
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