Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Three-dimensional Super-resolution Optical Fluctuation Imaging

Super-resolution optical fluctuation imaging (SOFI) achieves three-dimensional super-resolution by computing higher-order spatio-temporal cross-cumulants of stochastically blink-ing fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders. We present a balanced SOFI algorithm for mapping molecular parameters and for linearizing the brightness response and we outline a MATLAB toolbox for two- and three-dimensional SOFI analysis. We show super-resolved three-dimensional cell structures imaged with a multi-plane wide-field microscope. The simultaneous acqui-sition of several focal planes significantly reduces the acquisition time and helps limiting the photo-bleaching of the marker fluorophores

Photonic mode density effects on single-molecule fluorescence blinking

We investigated the influence of the photonic mode density (PMD) on the triplet dynamics of individual chromophores on a dielectric interface by comparing their response in the presence and absence of a nearby gold film. Lifetimes of the excited singlet state were evaluated in ordet to measure directly the PMD at the molecules position. Triplet state lifetimes were simultaneously determined by statistical analysis of the detection time of the fluorescence photons. The observed singlet decay rates are in agreement with the predicted PMD for molecules with different orientations. The triplet decay rate is modified in a fashion correlated to the singlet decay rate. These results show that PMD engineering can lead to an important suppression of the fluorescence, introducing a novel aspect of the physical mechanism to enhance fluorescence intensity in PMD-enhancing systems such as plasmonic devices

Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI)

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wide range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders for improving the resolution. Balanced super-resolution optical fluctuation imaging (bSOFI) analyses several cumulant orders for extracting molecular parameter maps, such as molecular state lifetimes, concentration and brightness distributions of fluorophores within biological samples. Moreover, the estimated blinking statistics are used to balance the image contrast, i.e. linearize the brightness and blinking response and to obtain a resolution improving linearly with the cumulant order. Using a widefield total-internal-reflection (TIR) fluorescence microscope, we acquired image sequences of fluorescently labelled microtubules in fixed HeLa cells. We demonstrate an up to five-fold resolution improvement as compared to the diffraction-limited image, despite low single-frame signal-to-noise ratios. Due to the TIR illumination, the intensity profile in the sample decreases exponentially along the optical axis, which is reported by the estimated spatial distributions of the molecular brightness as well as the blinking on-ratio. Therefore, TIR-bSOFI also encodes depth information through these parameter maps. bSOFI is an extended version of SOFI that cancels the nonlinear response to brightness and blinking heterogeneities. The obtained balanced image contrast significantly enhances the visual perception of super-resolution based on higher-order cumulants and thereby facilitates the access to higher resolutions. Furthermore, bSOFI provides microenvironment-related molecular parameter maps and paves the way for functional super-resolution microscopy based on stochastic switching

Noble-metal nanoparticles produced with colloidal lithography: fabrication, optical properties and applications

In this work, metal nanoparticles produced by nanosphere lithography were studied in terms of their optical properties (in connection to their plasmon resonances), their potential application in sensing platforms - for thin layer sensing and bio-recognition events -, and for a particular case (the nanocrescents), for enhanced spectroscopy studies. The general preparation procedures introduced early in 2005 by Shumaker-Parry et al. to produce metallic nanocrescents were extended to give rise to more complex (isolated) structures, and also, by combining colloidal monolayer fabrication and plasma etching techniques, to arrays of them. The fabrication methods presented in this work were extended not only to new shapes or arrangements of particles, but included also a targeted surface tailoring of the substrates and the structures, using different thiol and silane compounds as linkers for further attachment of, i.e. polyelectrolyte layers, which allow for a controlled tailoring of their nanoenvironment. The optical properties of the nanocrescents were studied with conventional transmission spectroscopy; a simple multipole model was adapted to explain their behaviour qualitatively. In terms of applications, the results on thin film sensing using these particles show that the crescents present an interesting mode-dependent sensitivity and spatial extension. Parallel to this, the penetrations depths were modeled with two simplified schemes, obtaining good agreement with theory. The multiple modes of the particles with their characteristic decay lengths and sensitivities represent a major improvement for particle-sensing platforms compared to previous single resonance systems. The nanocrescents were also used to alter the emission properties of fluorophores placed close to them. In this work, green emitting dyes were placed at controlled distances from the structures and excited using a pulsed laser emitting in the near infrared. The fluorescence signal obtained in this manner should be connected to a two- photon processes triggered by these structures; obtaining first insight into plasmon-mediated enhancement phenomena. An even simpler and faster approach to produce plasmonic structures than that for the crescents was tested. Metallic nanodiscs and nanoellipses were produced by means of nanosphere lithography, extending a procedure reported in the literature to new shapes and optical properties. The optical properties of these particles were characterized by extinction spectroscopy and compared to results from the literature. Their major advantage is that they present a polarization-dependent response, like the nanocrescents, but are much simpler to fabricate, and the resonances can be tailored in the visible with relative ease. The sensing capabilities of the metallic nanodiscs were explored in the same manner as for the nanocrescents, meaning their response to thin layers and to bio-recognition events on their surface. The sensitivity of these nanostructures to thin films proved to be lower than that of the crescents, though in the same order of magnitude. Experimental information about the near field extension for the Au nanodiscs of different sizes was also extracted from these measurements. Further resonance-tailoring approaches based on electrochemical deposition of metals on the nanodiscs were explored, as a means of modifying plasmon resonances by changing surface properties of the nanoparticles. First results on these experiments would indicate that the deposition of Ag on Au on a submonolayer coverage level can lead to important blue-shifts in the resonances, which would open a simple way to tailor resonances by changing material properties in a local manner

Tuning resonances on crescent-shaped noble-metal nanoparticles

The geometry of crescent-shaped noble-metal nanoparticles is systematically varied in terms of shape and size. The resulting changes in the plasmonic resonances of these structures are investigated by extinction spectroscopy revealing a rich polarization-dependent response in the near- infrared region of the electromagnetic spectrum. A first approach towards the understanding of this behaviour, in analogy to previous models on confined modes in nanometric metal slabs, is presented and discussed. Variations in several geometrical parameters lead to changes in the optical response that can be understood within this model. Qualitative changes in the response are seen at the transition of the structures from an open ‘crescent’ to a fully connected ring, pointing to a high field localization between the two tips of the structure

Controlled Polyelectrolyte Coating of Glass-Supported Metal Nanostructures

We investigated the coating of glass-supported gold nanostructures with polyelectrolyte multilayers. To achieve selective coating of either the metal or the glass, thiol and silane monolayers with different functionalities were used. Self-assembled monolayers of an oligo-(ethylene glycol)-terminated silane on glass allow for complete passivation and fully selective deposition of the polyelectrolytes on the metallic structures. This glass passivation forces more material on the gold than without use of this silane. Gold passivation was achieved on Au(111) but was not successful on the metallic nanostructures, giving rise to polyelectrolytes irregularly deposited on the nanoparticles in the form of blobs

Thin Layer Sensing with Multipolar Plasmonic Resonances

Gold crescent-shaped nanoparticles, exhibiting multiple resonances in the visible and near-infrared spectral region, were characterized in terms of their optical response to thin dielectric coatings. A strong red shift of the resonances is observed upon the addition of material. The coating thickness at which saturation of the shift sets in is resonance dependent and is directly connected to the spatial extension of the near field associated to each of the plasmonic modes, which is estimated through simplified theoretical models. These structures are advantageous for sensing for two reasons. First, the small modal volume makes them selectively sensitive to the first 10-30 nm distance from the gold. Second, each mode yields independent information about the coating and therefore in principle allows the determination of more than one free parameter of the coating, for example, thickness and refractive index

Three-dimensional Nano-Localization of Fluroescent Emitters

The use of fluorescence light for far-field imaging at the nanometer scale has drawn much attention worldwide. Example techniques include STED microscopy and localization microscopy (PALM, STORM). Typically, nanoscopy approaches have been focused on the manipulation and detection of the intensity of fluorescence light leading to lateral accuracy of a few nanometers. However, three-dimensional nanometer accuracy, in particular axial accuracy of the same order, remains a challenge. The phase of fluorescence light has shown to be valuable for axial measurements at the nano-scale. We extent the framework of phase microscopy to fluorescence light at the single-molecule level and demonstrate an approach combining super resolution microscopy with fluorescence interferometry for localizing a single nano-object to within several nanometres in all three dimensions. In a first proof of principle we demonstrate three-dimensional nano-localization of quantum dots within a standard deviation for the lateral and axial localization in the order of 6 nm and 4 nm, respectively. As the localization precision of the single nano-object in all three dimensions depends on the number of detected photons, we are exploring ways to increase the number of detected photons. As a result, we expect our approach to pave the way for three-dimensional, molecular-resolution fluorescence imaging

Three-dimensional nano-localization of single fluorescent emitters

We present a combination of self-interference microscopy with lateral super-resolution microscopy and introduce a novel approach for localizing a single nano-emitter to within a few nanometers in all three dimensions over a large axial range. We demonstrate nanometer displacements of quantum dots placed on top of polymer bilayers that undergo swelling when changing from an air to a water environment, achieving standard deviations below 10 nm for axial and lateral localization