Mitochondrial Cristae Revealed with Focused Light

Schmidt, Rupert; Wurm, Christian A.; Punge, Annedore; Egner, Alexander; Jakobs, Stefan; Hell, Stefan W.

doi:10.1021/nl901398t

Cited by 144 publications

(113 citation statements)

References 12 publications

(16 reference statements)

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“…2d), and imaged the vesicles using a newly developed improvement of stimulated emission depletion microscopy, isoSTED 12 , which allows a three-dimensional resolution on the size of single synaptic vesicles (~40-50 nm). Many small vesicle-sized spots could be identified (Fig.…”

mentioning

confidence: 99%

The same synaptic vesicles drive active and spontaneous release

Groemer²,

2010

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Synaptic vesicles release neurotransmitter both actively (upon stimulation) and spontaneously (at rest). It has been long assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin), in neuromuscular preparations from Drosophila, frog and mouse as well as rat cultured neurons, we suggest that the same vesicles participate in active and spontaneous release.1 Synaptic vesicles fuse with the neuronal plasma membrane to release their neurotransmitter contents both upon the arrival of action potentials (active release) and at rest (spontaneous release). It has been assumed for almost six decades that identical vesicles exocytose in both cases, an assumption which has been used to generate the quantal (vesicular) release theory 1 . This assumption is in agreement with the fact that prolonged active 2 or spontaneous release 3 can both release essentially all vesicles (see also 4,5 ). The issue of whether the same vesicles can be released both spontaneously and actively has been tested recently by investigating the loading and unloading of styryl (FM) dyes during vesicle recycling 6 . In cultured hippocampal neurons, the vesicles loaded with FM dyes during active or spontaneous recycling were unloaded efficiently only by the same release paradigm. Also, inhibiting synaptic vesicle re-acidification (and therefore refilling with neurotransmitter) by use of folimycin during spontaneous release specifically depleted the spontaneous pool 6 . Surprisingly, both of these findings have been later contested in similar experiments -FM dyes taken up either at rest or during stimulation could be released with identical kinetics, and the inhibition of reacidification during depolarization led to a cessation of spontaneous release 7 (see also 5 ). However, later studies have again found different FM dye release kinetics when using different loading paradigms 8,9 , and labeling experiments using the expression of a biotinylated variant of the synaptic vesicle marker VAMP2 (synaptobrevin) also supported the hypothesis that spontaneously and actively recycling vesicles are different 10 .We tested here this controversial issue by combining several fluorescence imaging assays. We sought to reproduce the FM dye loading/unloading paradigms used in the past. A number of FM dyes have been used, including FM 1-43 and FM 2-10 6-9 ; both dyes have provided evidence for a difference between the spontaneously and actively recycling vesicles 8,9 . It has been recently suggested that FM 2-10 reports this difference better than FM 1-43 9 . However, as FM 2-10 is less bright when compared to FM 1-43, it is typically used at very high concentrations (400 µM; ~4-fold higher than its membrane dissociation constant 11 ). Since the FM dyes have a detergent-like structure, they inhibit vesicle recycling at high concentrations 11 . We therefore decided to employ FM 1-43 in our work (the concentration typically used,...

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confidence: 99%

The same synaptic vesicles drive active and spontaneous release

Groemer²,

2010

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“…Scanning the excitation and the STED beam across the sample generates a sub-diffraction image. The spatial resolution of the final image depends on the intensity of the STED beam [1] , and spatial resolution as high as 30 nm has been obtained in biological samples [11] . The temporal resolution in STED is determined by the speed at which the focal spot can be scanned across the sample and the imaging area.…”

Section: Stimulated Emission Depletion -Stedmentioning

confidence: 99%

Super‐Resolution Microscopy: Going Live and Going Fast

Lakadamyali

2013

ChemPhysChem

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“…The depletion beam shrinks the effective size of the excitation spot to below the diffraction limit, theoretically as small as desired but practically limited to the same range as localization microscopy, 50±30 nm. STED and 4pi microscopy can be combined to enable isotropic multiwavelength resolution of approximately 30 nm in both the lateral and axial directions (Schmidt et al 2009). Multicolor 4pi-STED could potentially acquire images of the SC on the same size scale as localization microscopy.…”

Section: Other Superresolution Techniques Applicable To Meiotic Chrommentioning

confidence: 99%

Application of advanced fluorescence microscopy to the structure of meiotic chromosomes

Carlton

2013

Biophys Rev

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Chromosomes undergoing meiosis are defined by a macromolecular protein assembly called the synaptonemal complex which holds homologs together and carries out important meiotic functions. By retaining the molecular specificity, multiplexing ability, and in situ imaging capabilities of fluorescence microscopy, but with vastly increased resolution, 3D-SIM and other superresolution techniques are poised to make significant discoveries about the structure and function of the synaptonemal complex. This review discusses recent developments in this field and poses questions approachable with current and future technology.

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Mitochondrial Cristae Revealed with Focused Light

Cited by 144 publications

References 12 publications

The same synaptic vesicles drive active and spontaneous release

The same synaptic vesicles drive active and spontaneous release

Super‐Resolution Microscopy: Going Live and Going Fast

Application of advanced fluorescence microscopy to the structure of meiotic chromosomes

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