Sperm Gatekeeping: 3D Imaging Reveals a Constricted Entrance to Zebra Finch Sperm Storage Tubules.

Females across many internally fertilizing taxa store sperm, often in specialized storage organs in their reproductive tracts. In birds, several hundred sperm storage tubules exist in the utero-vaginal junction of the oviduct, and there is growing evidence that sperm storage in these tubules is selective. The mechanisms underlying female sperm storage in birds remain unknown because of our limited ability to make three-dimensional, live observations inside the large, muscular avian oviduct. Here, we describe a new application of fluorescence selective plane illumination microscopy to optically section oviduct tissue from zebra finch Taeniopygia guttata females label free by harnessing tissue autofluorescence. Our data provide the first description of the three-dimensional structure of sperm storage organs in any vertebrate to the best of our knowledge and reveal the presence of gate-like constricted openings that may play a role in sperm selection.

A trade-off between thickness and length in the zebra finch sperm mid-piece.

The sperm mid-piece has traditionally been considered to be the engine that powers sperm. Larger mid-pieces have therefore been assumed to provide greater energetic capacity. However, in the zebra finch Taeniopygia guttata, a recent study showed a surprising negative relationship between mid-piece length and sperm energy content. Using a multi-dimensional approach to study mid-piece structure, we tested whether this unexpected relationship can be explained by a trade-off between mid-piece length and mid-piece thickness and/or cristae density inside the mitochondrial helix. We used selective plane illumination microscopy to study mid-piece structure from three-dimensional images of zebra finch sperm and used high-resolution transmission electron microscopy to quantify mitochondrial density. Contrary to the assumption that longer mid-pieces are larger and therefore produce or contain a greater amount of energy, our results indicate that the amount of mitochondrial material is consistent across mid-pieces of varying lengths, and longer mid-pieces are simply proportionately 'thinner'.

Repurposing a photosynthetic antenna protein as a super-resolution microscopy label.

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd 1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.

From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups.

We show that sequential protein deposition is possible by photodeprotection of films formed from a tetraethylene-glycol functionalized nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (NPEOC-APTES). Exposure to near-UV irradiation removes the protein-resistant protecting group, and allows protein adsorption onto the resulting aminated surface. The protein resistance was tested using proteins with fluorescent labels and microspectroscopy of two-component structures formed by micro- and nanopatterning and deposition of yellow and green fluorescent proteins (YFP/GFP). Nonspecific adsorption onto regions where the protecting group remained intact was negligible. Multiple component patterns were also formed by near-field methods. Because reading and writing can be decoupled in a near-field microscope, it is possible to carry out sequential patterning steps at a single location involving different proteins. Up to four different proteins were formed into geometric patterns using near-field lithography. Interferometric lithography facilitates the organization of proteins over square cm areas. Two-component patterns consisting of 150 nm streptavidin dots formed within an orthogonal grid of bars of GFP at a period of ca. 500 nm could just be resolved by fluorescence microscopy.

A Novel Application of Non-Destructive Readout Technology to Localisation Microscopy.

The fitting precision in localisation microscopy is highly dependent on the signal to noise ratio. To increase the quality of the image it is therefore important to increase the signal to noise ratio of the measurements. We present an imaging system for localisation microscopy based on non-destructive readout camera technology that can increase the signal to noise ratio of localisation based microscopy. This approach allows for much higher frame rates through subsampling a traditional camera frame. By matching the effective exposure to both the start time and duration of a single molecule we diminish the effects of read noise and temporal noise. We demonstrate the application of this novel method to localisation microscopy and show both an increase in the attainable signal to noise ratio of data collection and an increase in the number of detected events.

Spatially modulated structural colour in bird feathers.

Eurasian Jay (Garrulus glandarius) feathers display periodic variations in the reflected colour from white through light blue, dark blue and black. We find the structures responsible for the colour are continuous in their size and spatially controlled by the degree of spinodal phase separation in the corresponding region of the feather barb. Blue structures have a well-defined broadband ultra-violet (UV) to blue wavelength distribution; the corresponding nanostructure has characteristic spinodal morphology with a lengthscale of order 150 nm. White regions have a larger 200 nm nanostructure, consistent with a spinodal process that has coarsened further, yielding broader wavelength white reflectance. Our analysis shows that nanostructure in single bird feather barbs can be varied continuously by controlling the time the keratin network is allowed to phase separate before mobility in the system is arrested. Dynamic scaling analysis of the single barb scattering data implies that the phase separation arrest mechanism is rapid and also distinct from the spinodal phase separation mechanism i.e. it is not gelation or intermolecular re-association. Any growing lengthscale using this spinodal phase separation approach must first traverse the UV and blue wavelength regions, growing the structure by coarsening, resulting in a broad distribution of domain sizes.

Interference lithographic nanopatterning of plant and bacterial light-harvesting complexes on gold substrates.

We describe a facile approach for nanopatterning of photosynthetic light-harvesting complexes over macroscopic areas, and use optical spectroscopy to demonstrate retention of native properties by both site-specifically and non-specifically attached photosynthetic membrane proteins. A Lloyd's mirror dual-beam interferometer was used to expose self-assembled monolayers of amine-terminated alkylthiolates on gold to laser irradiation. Following exposure, photo-oxidized adsorbates were replaced by oligo(ethylene glycol) terminated thiols, and the remaining intact amine-functionalized regions were used for attachment of the major light-harvesting chlorophyll-protein complex from plants, LHCII. These amine patterns could be derivatized with nitrilotriacetic acid (NTA), so that polyhistidine-tagged bacteriochlorophyll-protein complexes from phototrophic bacteria could be attached with a defined surface orientation. By varying parameters such as the angle between the interfering beams and the laser irradiation dose, it was possible to vary the period and widths of NTA and amine-functionalized lines on the surfaces; periods varied from 1200 to 240 nm and linewidths as small as 60 nm (λ/4) were achieved. This level of control over the surface chemistry was reflected in the surface topology of the protein nanostructures imaged by atomic force microscopy; fluorescence imaging and spectral measurements demonstrated that the surface-attached proteins had retained their native functionality.

Reversible switching between nonquenched and quenched states in nanoscale linear arrays of plant light-harvesting antenna complexes.

A simple and robust nanolithographic method that allows sub-100 nm chemical patterning on a range of oxide surfaces was developed in order to fabricate nanoarrays of plant light-harvesting LHCII complexes. The site-specific immobilization and the preserved functionality of the LHCII complexes were confirmed by fluorescence emission spectroscopy. Nanopatterned LHCII trimers could be reversibly switched between fluorescent and quenched states by controlling the detergent concentration in the imaging buffer. A 3-fold quenching of the average fluorescence intensity was accompanied by a decrease in the average (amplitude-weighted) fluorescence lifetime from approximately 2.24 ns to approximately 0.4 ns, attributed to the intrinsic ability of LHCII to switch between fluorescent and quenched states upon changes in its conformational state. The nanopatterning methodology was extended by immobilizing a second protein, the enhanced green fluorescent protein (EGFP), onto LHCII-free areas of the chemically patterned surfaces. This very simple surface chemistry, which allows simultaneous selective immobilization and therefore sorting of the two types of protein molecules on the surface, is a key underpinning step toward the integration of LHCII into switchable biohybrid antenna constructs.

Molecular weight dependent vertical composition profiles of PCDTBT:PC71BM blends for organic photovoltaics.

We have used Soxhlet solvent purification to fractionate a broad molecular weight distribution of the polycarbazole polymer PCDTBT into three lower polydispersity molecular weight fractions. Organic photovoltaic devices were made using a blend of the fullerene acceptor PC71BM with the molecular weight fractions. An average power conversion efficiency of 5.89% (peak efficiency of 6.15%) was measured for PCDTBT blend devices with a number average molecular weight of Mn = 25.5 kDa. There was significant variation between the molecular weight fractions with low (Mn = 15.0 kDa) and high (Mn = 34.9 kDa) fractions producing devices with average efficiencies of 5.02% and 3.70% respectively. Neutron reflectivity measurements on these polymer:PC71BM blend layers showed that larger molecular weights leads to an increase in the polymer enrichment layer thickness at the anode interface, this improves efficiency up to a limiting point where the polymer solubility causes a reduction of the PCDTBT concentration in the active layer.

Comparison of the physical characteristics of chlorosomes from three different phyla of green phototrophic bacteria.

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered "Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum" were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm(3) volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm(3)) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm(3)). Reflecting the contributions of thousands of pigment-pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000-2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.

Highly confined surface imaging by solid immersion total internal reflection fluorescence microscopy.

We report the use of a high-refractive-index aplanatic solid immersion lens (ASIL) in total internal reflection fluorescence (TIRF) microscopy. This new solid immersion total internal reflection fluorescence (SITIRF) microscopy allows highly confined surface imaging with a significantly reduced imaging depth compared with conventional TIRF microscopy. We explore the application of a high refractive index, low optical dispersion material zirconium dioxide in the SITIRF microscope and also introduce a novel system design which enables the SITIRF microscope to work either in the epi-fluorescence or TIRF modes with variable illumination angles. We use both synthetic and biological samples to demonstrate that the imaging depth in the SITIRF microscope can be confined to a few tens of nanometers. SITIRF microscopy has the advantages of performing highly selective imaging and high-resolution high-contrast imaging. Potential applications in biological imaging and future developments of SITIRF microscopy are proposed.

Synthesis and self-assembly of an amphiphilic poly(phenylene ethynylene) ionomer.

We have synthesized a conjugated amphiphilic polyelectrolyte, a poly(phenylene ethynylene) (PPE), and the structurally analogous neutral polymer. The solution-phase aggregation of the uncharged PPE can be reversibly controlled by varying the solvent polarity and concentration, while the charged polymer appears to self-assemble at any concentration in compatible solvents. These conclusions are based on a combination of absorption and photoluminescence spectroscopy and dynamic light scattering. Photoinduced absorption spectroscopy was also employed to investigate interchain electronic communication and the photoinduced production of free charge carriers. The uncharged PPE had a relatively high polaron yield, indicating pi-stacking of adjacent PPE chains and efficient exciton splitting, while the charged polymer did not produce polarons, indicating that the polymers are not pi-stacked despite their tendency to form aggregates. This is most likely due to the presence of the cationic trimethylammonium side chains which force neighboring polymer chains too far apart to achieve effective pi-orbital overlap. Polarons were observed in both polymers after chemical doping with iodine. The ability to control aggregation and interchain electronic communication could be a useful tool in designing nanostructured electronic materials.

Hexagonal nanoporous germanium through surfactant-driven self-assembly of Zintl clusters.

Surfactant templating is a method that has successfully been used to produce nanoporous inorganic structures from a wide range of oxide-based material. Co-assembly of inorganic precursor molecules with amphiphilic organic molecules is followed first by inorganic condensation to produce rigid amorphous frameworks and then, by template removal, to produce mesoporous solids. A range of periodic surfactant/semiconductor and surfactant/metal composites have also been produced by similar methods, but for virtually all the non-oxide semiconducting phases, the surfactant unfortunately cannot be removed to generate porous materials. Here we show that it is possible to use surfactant-driven self-organization of soluble Zintl clusters to produce periodic, nanoporous versions of classic semiconductors such as amorphous Ge or Ge/Si alloys. Specifically, we use derivatives of the anionic Ge9(4-) cluster, a compound whose use in the synthesis of nanoscale materials is established. Moreover, because of the small size, high surface area, and flexible chemistry of these materials, we can tune optical properties in these nanoporous semiconductors through quantum confinement, by adsorption of surface species, or by altering the elemental composition of the inorganic framework. Because the semiconductor surface is exposed and accessible in these materials, they have the potential to interact with a range of species in ways that could eventually lead to new types of sensors or other novel nanostructured devices.

Controlling optical properties and interchain interactions in semiconducting polymers by encapsulation in periodic nanoporous silicas with different pore sizes.

The photophysics of MEH-PPV incorporated into the pores of periodic silica hosts has been investigated in an effort to understand the role played by interchain aggregation and chain morphology in polaron production. In this work, guest/host interactions were used to incorporate MEH-PPV into the straight, homogeneous pores of hexagonal surfactant- or polymer-templated mesoporous silicas of varying pore diameters. Polarized photoluminescence and photoluminescence excitation spectroscopy were then used to investigate the polymers' environment within the silica pores. Experiments exploiting luminescence peak shifts and depolarization indicate that depending on the pore size and preparation conditions, the alignment and packing of the polymer chains within the pores could be controlled. Samples could be produced with isolated chains, interacting straight chains, and coiled interacting chains. The sub-bandgap absorption by polarons was then measured with photoinduced absorption as a function of pore size. Small-diameter pores that allowed single polymer chains to reside within the pore showed little evidence of interchain contact and had a low polaron yield. Increasing the number of polymer chains within the pore increased the polaron yield. Finally, when the pores were large enough that the chains could coil, strong polaron absorption was observed, indicative of a further increase in polaron yield or an increase in polaron lifetime. The polaron absorption spectra also sharpen and red shift with increasing pore diameter, suggesting that excitons may migrate to lower energy polymer segments in samples where polymer chains are both coiled and interacting.