Nanometer Axial Resolution by Three-Dimensional Supercritical Angle Fluorescence Microscopy

Winterflood, Christian M.; Ruckstuhl, Thomas; Verdes, Dorinel; Seeger, Stefan

doi:10.1103/physrevlett.105.108103

Cited by 58 publications

(50 citation statements)

References 23 publications

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“…37 By combination of the fluorescence intensities measured with these two channels, UAF and SAF, surface images with height information of subdiffraction z-resolution can be generated, a technique that was recently introduced as 3D-SAF microscopy. 38 The strength of 3D-SAF microscopy was exploited to distinguish surface features with different extensions in the zdirection. The 3D-SAF measurements reveal that in addition to the persistently surface bound aggregates observed so far, extended surface-tethered structures protruding between 1 and 3 μm or even deeper into the solution were present (Figure 3).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

Rabe

Soragni

Reynolds

et al. 2013

ACS Chem. Neurosci.

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The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

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Section: ■ Results and Discussionmentioning

confidence: 99%

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

Rabe

Soragni

Reynolds

et al. 2013

ACS Chem. Neurosci.

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“…At the present this so called 3D-SAF-microscopy technique is amongst the highest in spatial resolution [243]. The application of such high resolution imaging techniques will certainly have a strong impact on the research in the field of protein adsorption in the nearer future.…”

Section: Fluorescence Detection Techniquesmentioning

confidence: 99%

Understanding protein adsorption phenomena at solid surfaces

Rabe

Verdes

Seeger

2011

Advances in Colloid and Interface Science

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1,344

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Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined.

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“…Where molecules are rapidly tumbling, the Airy disc remains a reasonable approximation [8]. -The information contained in the supercritical emission can be used to measure molecular orientations, distance from the surface, and thickness of intermediate layers [8,11]. -Near the surface, the majority of the emitted light is emitted into the glass, further enhancing the efficiency of detection of molecules in an objective TIRF configuration (see Section 3.4) [9].…”

Section: Emission Characteristicsmentioning

confidence: 99%

“…This is known as supercritical angle fluorescence or SAF [10,11]. -The point spread function of a single molecule with a fixed orientation will differ significantly from the classical Airy disc (see Section 3.6 and Fig.…”

Section: Emission Characteristicsmentioning

confidence: 99%

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Knight

2014

Pure and Applied Chemistry

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Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

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Nanometer Axial Resolution by Three-Dimensional Supercritical Angle Fluorescence Microscopy

Cited by 58 publications

References 23 publications

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

On-Surface Aggregation of α-Synuclein at Nanomolar Concentrations Results in Two Distinct Growth Mechanisms

Understanding protein adsorption phenomena at solid surfaces

Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

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