Highly efficient optical detection of surface-generated fluorescence

Enderlein, Jörg; Ruckstuhl, Thomas; Seeger, Stefan

doi:10.1364/ao.38.000724

Cited by 161 publications

(105 citation statements)

References 8 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…The optical set-up is inverse to that of TIRF with an excitation beam illuminating the sample perpendicularly and a collection of fluorescence light emitted into the supercritical angle of the substrate using a parabolic mirror objective. The SAF technology itself (2000) [238,239], a SAF sensing instrument (2003) [240], and also a SAF imaging instrument (2007) [241,242] were recently developed. The strength of the SAF technique to study protein adsorption phenomena was demonstrated in some recent publications [44,102,118,187] or TIRF set-ups and, more recently, also with the SAF optics [244].…”

Section: Fluorescence Detection Techniquesmentioning

confidence: 99%

Understanding protein adsorption phenomena at solid surfaces

Rabe

Verdes

Seeger

2011

Advances in Colloid and Interface Science

Self Cite

1,345

1,239

View full text Add to dashboard Cite

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.

show abstract

Section: Fluorescence Detection Techniquesmentioning

confidence: 99%

Understanding protein adsorption phenomena at solid surfaces

Rabe

Verdes

Seeger

2011

Advances in Colloid and Interface Science

Self Cite

1,345

1,239

View full text Add to dashboard Cite

show abstract

“…In the work presented here, this disadvantage was overcome using super critical angle fluorescence (SAF) microscopy, which excludes all fluorescence except that arising from within ~ 200 nm of the surface. 32,33 Thus the adsorption of α−Syn can be studied in real time with no need for washing steps to remove unbound protein from the bulk solution.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Membrane Interaction and Disruption by α-Synuclein

et al. 2011

Self Cite

View full text Add to dashboard Cite

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein -synuclein. There is increasing evidence suggesting that the aggregation of -synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of -synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of -synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing -synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross--sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo. AbstractParkinson`s disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-Synuclein. There is increasing evidence suggesting that the aggregation of α−Synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface sensitive imaging technique super critical angle fluorescence microscopy and Förster resonance energy transfer we report the direct observation of α−Synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α−Synuclein studied, namely the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α−Synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross β-sheet aggregates and damaged the membrane at sub-micromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble the yet poorly...

show abstract

“…[3][4][5] Furthermore, it has been shown that the fluorescence lifetime strongly depends on the position and orientation of the SM with respect to the boundaries of the film, an effect known as the influence of the electromagnetic boundary conditions (EBC). [6][7][8][9][10][11][12][13] This effect is especially important in very thin polymer films (<30 nm). Therefore, several groups have devoted efforts to determine the 3D orientation of a SM in thin polymer films.…”

mentioning

confidence: 99%

Fluorescence Lifetimes and Emission Patterns Probe the 3D Orientation of the Emitting Chromophore in a Multichromophoric System

et al. 2004

Self Cite

View full text Add to dashboard Cite

W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/journals/jacsat.The optoelectronic properties of dyes embedded in thin polymer films are currently under intense study due to the use of such materials in nanotechnology 1 and nano-electronics. 2 The fluorescence lifetime of a single fluorescent molecule (SM) embedded in a thin film can fluctuate as a direct manifestation of the SM nanoenvironment (e.g., as a result of polymer dynamics). [3][4][5] Furthermore, it has been shown that the fluorescence lifetime strongly depends on the position and orientation of the SM with respect to the boundaries of the film, an effect known as the influence of the electromagnetic boundary conditions (EBC). [6][7][8][9][10][11][12][13] This effect is especially important in very thin polymer films (<30 nm). Therefore, several groups have devoted efforts to determine the 3D orientation of a SM in thin polymer films. 14-17 Techniques such as annular illumination (probing the orientation of the absorption transition dipole) 15,16 and wide-field defocusing (probing the emission transition dipole moment) have been proposed and exploited. 17 In this Communication, we report on the fluorescence behavior of a single first generation multichromophoric dendrimer with four perylene imide chromophores at the rim (G1R4) embedded in a thin polymer film (Chart 1). 18,19 A model developed to describe energy transfer in this system 13 invokes energy hopping between the different chromophores. It was suggested that, at any moment in time, the emission of a single dendrimer originates from the chromophore that has the lowest energy (fluorescent trapping site) due to the inhomogeneous nature of the polymer matrix used for immobilization. 20 Due to polymer dynamics and/or consecutive photobleaching, each of the chromophores in the dendrimer can in time become the fluorescent trapping site. Since the chromophores in the dendrimer have a well-defined 3D orientation with respect to each other (tetrahedral for G1R4), probing the dipole orientation of the emitting chromophore of a single G1R4 molecule using the two above-mentioned complementary observables, namely emission patterns and the EBC effect on the fluorescence lifetime, should allow us to validate the hopping model.To do so, G1R4 dendrimers at single-molecule concentration were embedded in thin (10-25 nm) zeonex (poly(norbornene)) films. To probe the orientation of the emitting chromophore emission dipole, we used defocused (1 µm defocusing distance with respect to the glass-polymer interface) wide-field imaging. 6,17 On the basis of the characteristic intensity distribution of the defocused images, the determination of the emission dipole orientation becomes feasible. Böhmer et al. 17 provided exact wave-optical calculations of these defocused images. Upon comparing the experimental data with calculated results, we obtained the emission dipole orientation.The patterns shown in Figure 1 were observed sequentially during the indicated period. One sees that th...

show abstract

Highly efficient optical detection of surface-generated fluorescence

Cited by 161 publications

References 8 publications

Understanding protein adsorption phenomena at solid surfaces

Understanding protein adsorption phenomena at solid surfaces

Mechanism of Membrane Interaction and Disruption by α-Synuclein

Fluorescence Lifetimes and Emission Patterns Probe the 3D Orientation of the Emitting Chromophore in a Multichromophoric System

Contact Info

Product

Resources

About