We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO(3) show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments.

Research on synthetic delivery vectors is of major interest for cell imaging and manipulation, as they allow an efficient transfer of nucleic acids, therapeutic proteins or small drugs into the cells. We have developed a library of L-lysine analogues that allow for highly efficient gene delivery with low cytotoxicity. However little is known on the exact mechanism of uptake and the final intracellular destination of the synthetic carriers. Therefore we have developed a novel optical technique based on a modulated excitation allowing for intracellular imaging of the triplet-lifetime and -yield of fluorophores attached to the delivery vector. Both these parameters are highly dependant on the intracellular environment thus provide insight into the subcellular localization of the labelled carrier. The method combines high temporal and spatial resolution and is compatible with a multiplicity of fluorophores. We performed series of model experiments to compare the triplet lifetime and triplet yield behaviour during the natural uptake mechanism to a series of controlled conditions. The latter include microinjection of fluorescently labelled carriers directly into the cytoplasm and cell nucleus as well as in vitro measurements under conditions mimicking physiological, acidic, or DNA rich environments. To validate our technique the results from the triplet imaging were compared with two complementary methods: carrier localization by subcellular fractionation and confocal laser scanning microscopy. --- Reference --- Geissbuehler et al. Triplet imaging of Oxygen consumption during the contraction of a single smooth muscle cell (A7r5). Biophysical Journal (2010) vol. 98 (2) pp. 339-349

SNAP-tag: We introduce a photoswitchable O6-benzylguanine derivative and demonstrate its use for super-resolution microscopy of SNAP-tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP-tagged microtubule structures with ∼25 nm resolution. The described probe in combination with the versatile SNAP-tag labeling opens new possibilities for imaging biological structures at the nanoscale. The advent of optical technologies that are capable of imaging structures with a resolution well below the diffraction limit has provided new insights into biology.1 One particularly attractive approach for nanoscopy that is based on the high-precision localization of single emitters is stochastic optical reconstruction microscopy (STORM) and related techniques.2 STORM has been successfully used for the imaging of different biological structures with nanoscale resolution.2a, 2c, 3 It requires the use of photoswitchable or blinking probes. This can be realized with pairs of cyanine dyes,1c, 4–6 single dyes (i.e., as in dSTORM5 and fluorescent proteins, among others)6 or even in a multicolor fashion. These switches can be cycled between “on” and “off” states many times; this allows modulation of the fluorescence emission of individual fluorophores in time such that only an optically resolvable subset of fluorophores is activated at any moment. Nanoscale super resolution is then achieved by precise localization of single emitters across all acquired frames.1a, 1c, 3e The accuracy of STORM imaging relies on the attachment of the photoswitchable probe that is specific to the structure of interest (as well as certain other factors, see the Supporting Information). This has commonly been achieved by chemically labeling antibodies with pairs of cyanine dyes; the antibody then targets the photoswitchable probe to the structure of interest.1c, 3c, 3d However, this approach has a number of problems. First, for each structure of interest, monoclonal antibodies with high specificity and affinity are needed. Second, the random chemical labeling of the antibody with the photoswitchable probes leads to heterogeneous samples. Third, antibodies are large proteins of 150 kD; this results in an artificial increase in size of the imaged structures.4, 5, 7 In principle, these limitations could be addressed by using self-labeling proteins to introduce photoswitchable probes suitable for STORM.8 The SNAP-tag is a small and highly soluble protein of 20 kD, and this makes it an ideal candidate for the construction of fusion proteins for nanoscopy; an example of this is the recent application for the stimulated emission depletion (STED) approach.9 It has been previously demonstrated that SNAP-tag fusion proteins can be labeled with a wide variety of different benzylguanine (BG) derivatives in cells.10 The labeling is highly specific, fast, stoichiometric and quantitative. Furthermore, SNAP-tag labeling can be achieved after fixation of cells; this is a prerequisite for its compatibility with STORM. Here, we apply SNAP-tag in combination with a photoswitchable substrate for the imaging of microtubules with an estimated resolution of ∼25 nm (FWHM, see Figure S12). Specifically, we synthesized a probe that contains Cy3 and Cy5 dye molecules as well as a BG moiety (Scheme 1). The BG permits the targeting of the Cy3-Cy5 photoswitch to SNAP-tag fusion proteins. Photoswitchable probe BG-Cy3-Cy5 for labeling of SNAP-tag fusion proteins of interest (POI). We synthesized BG-Cy3-Cy5 in four steps from commercially available materials and demonstrated that the probe reacts with the SNAP-tag in vitro (Figures S1 and S5). We then attempted to use the probe for nanoscopy of microtubules, which are a copolymer of α- and β-tubulin.11 Towards this end, we expressed β-tubulin as a C-terminal fusion of SNAP-tag (β-tubulin-SNAP) in U2OS cells. To verify the applicability of BG-Cy3-Cy5 for the labeling of proteins in cells, we fixed U2OS cells expressing β-tubulin-SNAP and incubated them with the probe; this incubation was followed by a washing step. As a control, we performed α-tubulin immunostaining of the cells in parallel. Confocal fluorescence imaging demonstrates a highly specific labeling of β-tubulin-SNAP with Cy3-Cy5, as shown by the colocalization of the signals from the immunostaining of α-tubulin and the SNAP-tag labeling (Figure 1). It should also be noted that SNAP-tag remains active and can be labeled after methanol fixation, whereas green fluorescent protein (GFP) is known to lose fluorescence under similar conditions.12 Confocal fluorescence images of cells expressing β-tubulin–SNAP fusion protein. Overlapping of the fluorescence resulting from the chemical labeling and immunostaining indicates the correct localization of β-tubulin–SNAP to the microtubules and demonstrates the specificity of the approach. Cellular α-tubulin was stained using mouse anti-α-tubulin and Alexa 488 labeled anti-mouse IgG antibody (green). Transiently expressed β-tubulin–SNAP fusion protein was labeled with the SNAP-tag substrate BG-Cy3-Cy5 (red). Nuclear DNA staining with Hoechst 33342 was used as a reference (blue). Cells were fixed with methanol prior to staining. The labeled samples were imaged using a custom designed inverted total internal reflection (TIRF) microscope in epi-illumination. The samples were embedded in an imaging buffer containing mercaptoethanol and an oxygen scavenging system prior to imaging.2b Two laser excitation sources were used: a red HeNe laser (633 nm, 2.5 mW) for imaging and a green argon laser (514 nm, attenuated to 200 μW) for activating the photoswitchable tags (Figures S6 and S7). For the STORM image reconstruction, 5000 images were acquired at a 20 Hz frame rate. Reconstructed images after single molecule localization were filtered using an image processing freeware to remove the nonstructured background without any significant loss of information (see the Supporting Information). An example of a representative image analysis sequence is depicted in Figure 2. The improvement in resolution when comparing Figure 1 with 2 B and C is already evident; structures in the order of 40 nm become clearly resolvable (Figures 2, S10, and S11). The isolated spots, which are not part of any structure in the image, are most likely free labeled β-tubulin-SNAP or unreacted BG-Cy3-Cy5. The statistical analysis of the image shows a mean width (2σ) of approximately 36 nm (Figure 2 D). Stochastic Optical Reconstruction Microscopy of microtubules labeled with BG-Cy3-Cy5 by β-tubulin–SNAP. A) The wide field microscopy image of a selected cell (normalized color scale by the mean intensity of all frames); B) the complete field of view after STORM reconstruction; C) zoom into the microtubules highlighted in B)–white inset; D) transversal localization distribution on the microtubule fitted to Gaussian distribution. The localization distribution is averaged over a 200 nm large region along the structure (white box in C)). The Gaussian fit shows a mean width (2 s) of approximately 36 nm, a characteristic feature size well below the diffraction limit. The results presented in Figures 1 and 2 show the applicability of our new photoswitchable probes for the specific labeling of SNAP-tag fusion proteins in cells and the subsequent STORM-based nanoscopy. Microtubules visualized with β-tubulin-SNAP have been resolved well below the diffraction limit. The characteristic dimensions of these structures measured by this method are 40±10 nm in diameter; this value is close to the theoretical value of 30 nm, which is obtained from a microtubule diameter of ∼25 nm, and an estimated distance of ∼2.5 nm from the N terminus of the SNAP-tag to its reactive cysteine residue. It is noteworthy that this measured size of the microtubule is smaller than that measured with STORM based on antibody staining (60 nm).2b An additional feature of the chemical labeling is that the fixed stoichiometry of one label per protein permits an estimation of the number of labeled proteins incorporated into the structure of interest. In summary, we have introduced a targeted photoswitchable probe for the labeling of SNAP-tag fusion proteins in cells and have successfully used them for STORM-based nanoscopy. The small size of the SNAP-tag and the simplicity of its labeling with a large variety of fluorescent probes make it well suited for the imaging of biological structures. Furthermore, it should be straightforward to extend the approach described here to multicolor STORM, or dSTORM,5 using different, “orthogonal” self-labeling tags, such as the CLIP-tag.10a We therefore believe that the approach described will significantly broaden the possibilities of performing nanoscopy of biological structures. The authors acknowledge the funding support of the Swiss National Science Foundation (Synergia project CRSII3 125463; K-23K1-116242/1; 315200-116729). G.L. acknowledges the Federation of European Biochemical Societies for a long-term fellowship. The authors thank Francois Aguet and Pierre Gönczy for interesting and helpful discussions. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

The diffusion of charged proteins in liquid-filled nanometer-sized apertures with charged surfaces has been investigated with fluorescence correlation spectroscopy (FCS). Based on a two-dimensional (2D) multicomponent diffusion model, key parameters such as the number of molecules diffusing freely inside the nanochannel or interacting with the surfaces, together with the specific diffusion parameters, could be extracted. Different regimes of diffusion have been observed and described by a model, which takes into account the steric exclusion, the reversible surface adsorption of the biomolecules, and the exclusion-enrichment effect that is due to the charge of the proteins and the ionic strength of the solution. Conditions where the diffusion of proteins through nanoconfined spaces can be of the same magnitude as in the bulk were both predicted and experimentally verified.

Fluorescence localization microscopy (i.e., PALM, STORM) has enabled optical imaging at nanometer-scale resolutions. The localization algorithms used in these techniques rely on fitting a 2-D Gaussian to the in-focus image of individual fluorophores. For fixed fluorophores, however, the observed diffraction pattern depends on the orientation of the underlying molecular dipole and does not necessarily correspond to a section of the system's point spread function. By using a physically realistic image formation model for dipoles to perform the fit, both the position and orientation of the dipole can be estimated with high accuracy, improving upon Gaussian localization. In this paper, we present an algorithm for joint position and orientation estimation based on a 3-D steerable filter, and show that the results are near-optimal with respect to the Cramer-Rao bounds. We show that patterns generated using estimated positions and orientations closely fit experimental measurements.

Fluorophores that are fixed during image acquisition produce a diffraction pattern that is characteristic of the orientation of the fluorophore's underlying dipole.Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by applying Gaussian-based fitting algorithms to in-focus images of individual fluorophores; when applied to fixed dipoles, this can lead to a bias in the range of 5-20 nm.We introduce a method for the joint estimation of position and orientation of dipoles, based on the representation of a physically realistic image formation model as a 3-D steerable filter.Our approach relies on a single, defocused acquisition.We establish theoretical, localization-based resolution limits on estimation accuracy using Cramér-Rao bounds, and experimentally show that estimation accuracies of at least 5 nm for position and of at least 2 degrees for orientation can be achieved.Patterns generated by applying the image formation model to estimated position/orientation pairs closely match experimental observations.

With fluorescence correlation spectroscopy (FCS), the diffusion and concentration of proteins (wheat germ agglutinin) in a nanofluidic system are measured on a single molecule level. By increasing the ionic concentration, the Debye length of the electrical double layer (EDL) decreases altering the strongly enhanced surface interactions of charged proteins in nanochannels. A theoretical model of the transport of charged mole- cules in nanofluidics facilitates the design of future nanochannel applications.

Odorant receptors are an excellent example of natural superiority in specifically binding, specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02 mu m(-2)min(-1). The expression and incorporations of single OR5s were completed within about,8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

We present the development and application of a dual-color setup that can be used for confocal fluorescence correlation spectroscopy (FCS), total internal reflection FCS (TIR-FCS) as well as dual color imaging.As exemplary application we performed preliminary measure-ments for DNA-Sequencing with a novel surface chemistry. Motivation • Single molecule detection of chemical kinetics A(red)+B(blue) AB• Monitoring biological processes (eg molecular binding essays)• in vivo observations of dynamics of molecular motors Application functionalized surfaceLinker to surfacecircularized singleDNA strandconfined excitation anddetection Volumecompleted DNA strandEnzymefree Nucleotides DNA Sequencing • improved surface chemistry• bind single DNA strand to surface• labeling nucleotides• observation in confocal volume Figure 4: DNA Sequencing methodFigure 5: Image of dual colored spots afterseveral minutes of nucleotide-incorporation Outlook • Advanced concepts for further confined sampling volumes• Higher concentrations of labeled molecules• More colors or/and lifetime seperation• More biological systems

We present an imaging contrast based on triplet state life-time extracted from a series of images taken with modulated excitation schemes. This technique is validated by visualizing oxygen consumption dur- ing contraction of smooth muscle cells.