Hyperspectral super-resolution imaging with far-red emitting fluorophores using a thin-film tunable filter.

New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-color or hyperspectral SMLM can pose particular challenges which affect image quality and data interpretation, such as unequal photophysical performance of fluorophores and non-linear image registration issues, which arise as two emission channels travel along different optical paths to reach the detector. In addition, using evanescent-wave based approaches (Total Internal Reflection Fluorescence: TIRF) where beam shape, decay depth, and power density are important, different illumination wavelengths can lead to unequal imaging depth across multiple channels on the same sample. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images arising from fluorescence emission captured within ∼15 nm to 20 nm spectral windows (or detection bands). By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we investigated several far-red emitting fluorophores and identified an optimal fluorophore pair for two-color SMLM using this method. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20, and the peroxisomal protein, PMP70.

Single-molecule localization microscopy of septin bundles in mammalian cells.

Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes, including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified Saccharomyces cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a direct stochastic optical reconstruction microscopy and photoactivated localization microscopy study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero-octamers and an approximate 30-35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9.

Nanoscale Pulling of Type IV Pili Reveals Their Flexibility and Adhesion to Surfaces over Extended Lengths of the Pili.

Type IV pili (T4P) are very thin protein filaments that extend from and retract into bacterial cells, allowing them to interact with and colonize a broad array of chemically diverse surfaces. The physical aspects that allow T4P to mediate adherence to many different surfaces remain unclear. Atomic force microscopy (AFM) nanoscale pulling experiments were used to measure the mechanical properties of T4P of a mutant strain of Pseudomonas aeruginosa PAO1 unable to retract its T4P. After adhering bacteria to the end of an AFM cantilever and approaching surfaces of mica, gold, or polystyrene, we observed adhesion of the T4P to all of the surfaces. Pulling of single and multiple T4P on retraction of the cantilever from the surfaces could be described using the worm-like chain (WLC) model. Distinct peaks in the measured distributions of the best-fit values of the persistence length Lp on two different surfaces provide strong evidence for close-packed bundling of very flexible T4P. In addition, we observed force plateaus indicating that adhesion of the T4P to both hydrophilic and hydrophobic surfaces occurs along extended lengths of the T4P. These data shed new light, to our knowledge, on T4P flexibility and support a low-affinity, high-avidity adhesion mechanism that mediates bacteria-surface interactions.

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures-surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations.

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir-Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.

Using nanoscale substrate curvature to control the dimerization of a surface-bound protein.

The influence of surface geometry on adsorbed proteins offers new possibilities for controlling quaternary structure by manipulating protein-protein interactions at a surface, with applications that are relevant to protein aggregation, fibrillation, ligand binding, and surface catalysis. To understand the effect of surface curvature on the structure of the surface-bound protein ß-lactoglobulin (ß-LG), we have used a combination of polystyrene (PS) nanoparticles (NPs) and ultrathin PS films to fabricate chemically pure, hydrophobic surfaces that have nanoscale curvature and are stable in aqueous buffer. We have used single molecule force spectroscopy to measure the detachment contour lengths L(c) for ß-LG adsorbed on the highly curved PS surfaces, and we compare these values in situ to those measured for ß-LG adsorbed on flat PS surfaces on the same samples. The L(c) distributions measured on all flat PS surfaces show a large monomer peak near 60 nm and a smaller dimer peak at 120 nm. For 190 and 100 nm diameter NPs, which are effectively flat on the scale of the ß-LG molecules, there is no measurable difference between the L(c) distributions obtained for the flat and curved surfaces. However, for 60 nm diameter NPs the dimer peak is smaller, and for 25 nm diameter NPs the dimer peak is absent, indicating that the number of surface-bound dimers is significantly reduced by an increase in the curvature of the underlying surface. These results indicate that surface curvature provides a new method of manipulating protein-protein interactions and controlling the quaternary structure of adsorbed proteins.

Modified spin-coating technique to achieve directional colloidal crystallization.

Fabricating large single crystals with colloidal spheres as building blocks is challenging and of competitive interest. Spin-coating of colloids offers a robust technique, which is highly reproducible in obtaining colloidal crystals even at fast dynamical regimes; however, these crystals are intrinsically polycrystalline due to the axial symmetry of spin-coating. We report a new method that applies a nonuniform electric field during the spin-coating process. By arranging the field direction to be stationary in the rotating frame, we are able to break the axial symmetry and to orient the colloids along one predefined direction. By regulating the applied field strength, we demonstrate local control over the orientation of the crystallites, and thus, the orientation is determined by the applied field strength.