Spatiotemporal Heterogeneity of κ-Carrageenan Gels Investigated via Single-Particle-Tracking Fluorescence Microscopy

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions have remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ∼1-5 × 10-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ∼1 × 10-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from the diffusional data indicated that the dense network regions have a characteristic diameter of ∼1 μm and show mobility on the second-to-minute timescale. sptFM provides an unprecedented view of spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high-molecular weight solutes.</p

Pinpointing macromolecular motion : Single-particle tracking fluorescence microscopy advances and applications

Macromolecular motion is dictated by diffusion on &gt;100 nm spatial scales, while specific interactions occur on the nanoscale based on Van der Waals, electrostatic, and hydrophobic/hydrophilic forces. Characterising macromolecular motion reveals fundamental insights when relating diffusional behaviour to the function of a macromolecule of interest. To adequately investigate diffusion, a technique is required that ideally 1) minimises sample invasion and destruction; 2) minimises spatiotemporal averaging; 3) reaches molecular specificity; 4) is accessible by non-experts of the technique; and 5) has a spatiotemporal resolution of at least &sim;5-100 nm and &sim;10 ms, and preferably even better. Single-particle tracking fluorescence microscopy (sptFM) is a derivative of single-molecule localization microscopy (SMLM) and offers the possibility to investigate macromolecular motion while upholding these criteria. SMLM is a super-resolution optical microscopy methodology characterised by localizing point spread functions (PSFs) originating from single fluorescent molecules, with an accuracy surpassing the diffraction limit of light by roughly one order of magnitude. SptFM tracks single molecules moving through time, describing their position with &sim;5-40 nm spatial resolution and good temporal resolution. These motions can then be quantitatively characterised and used to reveal macromolecular behaviour. While sptFM is a promising technique to observe macromolecular motion, it can be further improved. SptFM can then be applied to study macromolecular diffusion in life science and soft matter. This thesis aims to advance the field of sptFM by increasing the achievable spatiotemporal resolution, and by increasing the accessibility of the hardware (miCube microscopy platform) and software (phasor-based single molecule localization microscopy or pSMLM) in sptFM. Next, sptFM, enriched by these advances, is applied to study dynamic CRISPR-Cas9 behaviour in vivo, and to study the spatiotemporal heterogeneity of &kappa;-carrageenan hydrogels

Spatiotemporal Heterogeneity of κ-Carrageenan Gels Investigated via Single-Particle-Tracking Fluorescence Microscopy

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions have remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ∼1-5 × 10-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ∼1 × 10-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from the diffusional data indicated that the dense network regions have a characteristic diameter of ∼1 μm and show mobility on the second-to-minute timescale. sptFM provides an unprecedented view of spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high-molecular weight solutes.</p

Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.</p

Hexakis(adamantyltrimethyl-ammonium) cyclooctasilicate tetratetracontahydrate

The title compound, 6C13H24N+· H2Si8O206-·44H2O, belongs to the class of cyclo-silicate hydrates, which structurally can be positioned between the zeosils and the clathrate hydrates. [Si8O 18(OH)2] cubes carrying six negative charges are located on crystallographic inversion centres and are surrounded by six adamantyl-trimethyl-ammonium cations. © 2010 International Union of Crystallography

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 106 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.ImPhys/Quantitative Imagin

Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.</p

Quantitative Structural Analysis of Fat Crystal Networks by Means of Raman Confocal Imaging

The techniques that are currently available to assess fat crystal networks are compromised with respect to invasive sample preparation and ability to quantify compositional and structural features. Raman confocal hyperspectral imaging coupled to analysis with multivariate curve resolution can address these bottlenecks, as it provides label-free, noninvasive chemical information in three dimensions (3D). We demonstrate the ability to acquire compositional maps of dispersions of micronized fat crystals (MFC) in oil, which contain local concentrations of liquid oil and solid fat with submicron spatial resolution and with acquisition times in the order of 10 min. From the compositional maps, we can derive quantitative information on the size and porosity of fat crystal flocs, as well as the solid fat content of the embedding continuous phase. Furthermore, the fractal dimension of the fat crystal network could be determined from the compositional maps via the box-counting method and via the porosities of the crystal flocs. This makes it feasible to assess the validity of the weak-link network theory under industrial relevant conditions. The confocal imaging mode allows for straightforward acquisition of 3D compositional cubes by recording a stack of two-dimensional (2D) images. The box-counting fractal dimension analysis performed on 2D maps can be extended to 3D cubes, which allows for straightforward verification that MFC networks are self-similar rather than self-affine

Chromate-mediated one-step quantitative transformation of PW 12 into P 2W 20 polyoxometalates

The addition of H 3PW 12O 40 to an aqueous solution of K 2CrO 4 led to the hydrolysis of [PW 12O 40] 3- with its quantitative conversion into K 13[KP 2W 20O 72]. K 13[KP 2W 20O 72] in these chromate-containing solutions was characterized by 183W and 31P NMR, EXAFS, SAXS, and EPR spectroscopy. © 2012 Wiley-VCH Verlag GmbH &amp; Co. KGaA, Weinheim