Neuro at the Nanoscale: Diffraction-Unlimited Imaging with STED Nanoscopy

Abstract

Recent breakthroughs in fluorescence microscopy have pushed spatial resolution well beyond the classical limit imposed by diffraction. As a result, the field of nanoscopy has emerged, and diffraction-unlimited resolution is becoming increasingly common in biomedical imaging applications. In this review, we recap the principles behind STED nanoscopy that allow imaging beyond the diffraction limit, and highlight both historical and recent advances made in the field of neuroscience as a result of this technology.

Keywords: Dendrite; Fluorescence; Nanoscopy; Neuroimaging; Neuron; Neurotransmitter Release; STED Microscopy; Spine; Super-resolution; Synapse.

Figure 1.

Comparison of confocal microscopy and STED nanoscopy. (A) Excitation and corresponding fluorescence profiles for a conventional confocal microscope. (B) The addition of a depletion focus confines fluorescence to a sub-diffraction-sized volume in STED nanoscopy. (C) Cartoon of dendritic spines and simulated (D) confocal and (E) STED images demonstrate the effect of enhanced spatial resolution in a fluorescence microscope. Scale (C–E) 500 nm.

Figure 2.

Principles of STED nanoscopy. (A) Jablonski diagram showing fluorophore transitions between the ground and excited states for excitation, fluorescence, and depletion. Resolution enhancement through targeted switching is achieved when fluorophores are forced out of the excited state by the depletion laser. Lateral (B) and axial (C) depletion profiles generated by a helical phase ramp (inset, B). Lateral (D) and axial (E) depletion profiles generated by central half-wave phase step (inset, D).

Figure 3.

An early result of STED microscopy, imaging the Drosophila active zone component bruchpilot [from (Kittel et al. 2006)]. Left: Confocal images of bruchpilot fluorescently labeled with the selective antibody Nc82. Whereas discrete protein complexes can be resolved, labeled domains are diffraction limited, and exhibit no discernible substructure. Middle: higher resolution STED images of the same field of view, revealing the same labeled domains to be toroid-shaped. Right: higher magnification of bruchpilot, showing single and multi-ring clusters. White arrows and arrowheads indicate single rings and multi-ring clusters, respectively. Red arrow indicates a protein cluster viewed parallel to the plane spanned by the synaptic cleft. Scale, 1 μm.

Figure 4.

Nanoscale changes in dendritic spine morphology evoked by focal uncaging of glutamate [after Fig. 6 of Tonnesen et al. (2014)]. (A) A single spine before (left) and after (right) induction of spine-specific long-term potentiation (LTP) by two-photon uncaging of glutamate (uLTP). Note the marked and highly resolved changes in spine head volume and spine neck length and width. (B) Enlargements of the boxed areas in (A). (C) Group data showing large, input-specific increases in spine volume following uLTP. Red trace shows head-volume changes in the stimulated spine. Black shows relative absence of volume changes in neighbor (unstimulated) spines, illustrating synapse specificity. Blue shows relative absence of uLTP in high magnesium, illustrating NMDA receptor dependence. (D) Group data showing decreases in spine-neck length following uLTP. Trace colors and corresponding conditions as in (C). (E) Group data showing increases in spine-neck width following uLTP. Trace colors and corresponding conditions as in (C). Scale bars, 500 nm. *P<0.05; **P<0.01; ***P< 0.001.

Figure 5.

Highly periodic arrangement of cytoskeletal components in neurites, observed at the nanoscale. (A, B) Taken from Xu et al. (2013); (C, D) taken from D’Este et al. (2015). (A) Ring-like arrangement of fluorescently labeled spectrin along the axon of a cultured hippocampal neuron. Inset shows a y-z cross-section of the boxed area. (B) Histogram of distances between spectrin rings, consistent with a highly repeated structural motif with ~180-nm spatial periodicity. (C, D) Images of sirActin-labeled axons (C) and dendrites (D) of cultured hippocampal neurons. Scale (C, D) 1 μm.