Real-time imaging of single synaptic vesicles in live neurons

doi:10.1007/s11515-016-1397-z

Front. Biol.

2016, Vol. 11

Issue (2) : 109-118 https://doi.org/10.1007/s11515-016-1397-z

REVIEW

Real-time imaging of single synaptic vesicles in live neurons

Chenglong Yu¹,Min Zhang¹,Xianan Qin²,Xiaofeng Yang¹,Hyokeun Park^1,^2,^3,^*(

)

1. Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
2. Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
3. State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

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Abstract

Recent advances in fluorescence microscopy have provided researchers with powerful new tools to visualize cellular processes occurring in real time, giving researchers an unprecedented opportunity to address many biological questions that were previously inaccessible. With respect to neurobiology, these real-time imaging techniques have deepened our understanding of molecular and cellular processes, including the movement and dynamics of single proteins and organelles in living cells. In this review, we summarize recent advances in the field of real-time imaging of single synaptic vesicles in live neurons.

Keywords single synaptic vesicle real-time imaging exocytosis tracking

Corresponding Author(s): Hyokeun Park

Just Accepted Date: 18 April 2016 Online First Date: 09 May 2016 Issue Date: 17 May 2016

Cite this article:

Chenglong Yu,Min Zhang,Xianan Qin, et al. Real-time imaging of single synaptic vesicles in live neurons[J]. Front. Biol., 2016, 11(2): 109-118.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-016-1397-z
https://academic.hep.com.cn/fib/EN/Y2016/V11/I2/109

Fig.1 Early examples of monitoring exocytosis of single synaptic vesicles. (A) Exocytosis of single vesicles loaded with FM 1-43 in presynaptic ribbon terminals. (A1) The experimental scheme. Total internal reflection fluorescence microscopy (TIRFM) is used to illuminate the sample. (A2) A newly arriving vesicle enters the active zone during stimulation and nearly fully releases FM 1-43. The open symbols correspond to the images shown above. The scale bar represents 0.5 microns. Modified from (Zenisek et al., 2000). (B) Two distinct fusion modes can be triggered by stimulation with a single action potential (AP). (B1) Images (above) and a corresponding time course showing full-collapse fusion triggered by a single AP. Note that the synaptic vesicle released its entire FM 1-43 contents in one step. (B2) Images and a corresponding time course showing kiss-and-run fusion triggered by a single AP. Note that the synaptic vesicle released only a fraction of its FM 1-43 contents. Modified from (Aravanis et al., 2003).

Fig.2 Kiss-and-run fusion and full-collapse fusion detected by the fluorescence change of a single quantum dot. (A) Kiss-and-run is revealed by a 15% increase in fluorescence intensity triggered by the transient opening of the fusion pore in a synaptic vesicle loaded with a single quantum dot (shown as an “uptick” in the red trace). (A1) Schematic diagram depicting the increase in fluorescence upon exposure to the extracellular solution (pH ~7.3). (A2) The change in the quantum dot’s fluorescence signal during kiss-and-run events (red arrows in the trace and red bars in the histogram) and upon full-collapse fusion (the blue arrow in the trace and the blue bars in the histogram). Modified from (Zhang et al., 2009). (B) Kiss-and-run fusion was detected by the partial quenching of fluorescence of a single quantum dot inside a vesicle. The images and corresponding fluorescence trace indicate that a single quantum dot residing inside a vesicle undergoing kiss-and-run fusion was partially quenched by trypan blue in the extracellular solution. During the kiss-and-run event, the fluorescence signal dropped partially (indicated by the red arrow), then was completely quenched upon full-collapse fusion (indicated by the black arrow). The scale bar represents 0.8 microns. Modified from (Park et al., 2012).

Fig.3 Tracking single synaptic vesicles. (A) Representative images of a synaptic vesicle loaded with a single quantum dot, a presynaptic terminal containing FM 4-64?labeled vesicles, and a postsynaptic compartment labeled with GFP-tagged PSD-95 (PSD-95-GFP). The graph below shows a three-dimensional trace of the quantum dot-loaded vesicle, showing that this vesicle moved from the center of the presynaptic terminal to the synaptic cleft, where it underwent exocytosis. The scale bar represents 0.8 microns. (B) Three-dimensional trace of one single synaptic vesicle that moved from one synapse to another synapse, and then underwent exocytosis. Modified from (Park et al., 2012). (C) Differential mobility of single synaptic vesicles labeled with the styryl dye SGC5 either by spontaneous activity or by evoked activity. (C1) Images and the corresponding trace of a single synaptic vesicle labeled by evoked activity. (C2) Images and the corresponding trace of a synaptic vesicle labeled by spontaneous activity. Vesicles labeled by evoked activity were more mobile than vesicles labeled by spontaneous activity. Modified from (Peng et al., 2012).

Fig.4 Figure box2 Point-spread functions (PSFs) of Cy3 labeled DNA. The expanded PSF had a width of 287 nm, photon counting of 14200 and signal to noise ratio of 32. The localization accuracy in the example was 1.3 nm. Modified from (Yildiz et al., 2003).

Fig.5 Figure box3 Schematics of stimulated emission depletion (STED) microscopy. The depletion laser creates a donut-like beam pattern using a phase mask and selectively suppresses fluorescent molecules at the periphery. Fluorescent molecules in the tiny central region are allowed to emit photons.

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