Multicolor super-resolution imaging with photo-switchable fluorescent probes

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with a spatial resolution of 20 to 50 nanometers. Multicolor super-resolution imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multicolor imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.     

Imaging the pore structure of geomaterials

Laser scanning confocal microscopy can be used to image the pore structure of geologic materials in three dimensions at a resolution of 200 nanometers. The technique involves impregnation of the void space with an epoxy doped with a fluorochrome whose fluorescent wavelength matches the excitation wavelength. Optical sections with a thickness of less than 1 micrometer can be sliced from thick polished sections and combined to produce three-dimensional reconstructions. Application of the technique to rocks with porosities from 1 to 20 percent reveals the geometric complexity of the pore space. The technique can also be applied to other brittle solids such as ceramics.     

Three-dimensional orientation mapping in the transmission electron microscope

Over the past decade, efforts have been made to develop nondestructive techniques for three-dimensional (3D) grain-orientation mapping in crystalline materials. 3D x-ray diffraction microscopy and differential-aperture x-ray microscopy can now be used to generate 3D orientation maps with a spatial resolution of 200 nanometers (nm). We describe here a nondestructive technique that enables 3D orientation mapping in the transmission electron microscope of mono- and multiphase nanocrystalline materials with a spatial resolution reaching 1 nm. We demonstrate the technique by an experimental study of a nanocrystalline aluminum sample and use simulations to validate the principles involved.     

Breaking the diffraction barrier: optical microscopy on a nanometric scale

In near-field scanning optical microscopy, a light source or detector with dimensions less than the wavelength (lambda) is placed in close proximity (lambda/50) to a sample to generate images with resolution better than the diffraction limit. A near-field probe has been developed that yields a resolution of approximately 12 nm ( approximately lambda/43) and signals approximately 10(4)- to 10(6)-fold larger than those reported previously. In addition, image contrast is demonstrated to be highly polarization dependent. With these probes, near-field microscopy appears poised to fulfill its promise by combining the power of optical characterization methods with nanometric spatial resolution.     

Atomic-resolution microscopy in water

The scanning tunneling microscope is revolutionizing the study of surfaces. In ultra-high vacuum it is capable not only of imaging individual atoms but also of determining energy states on an atom-by-atom basis. It is now possible to operate this instrument in water. Aqueous optical microscopy is confined to a lateral resolution limit of about 2000 angstroms, and aqueous x-ray microscopy has yielded a lateral resolution of 75 angstroms. With a scanning tunneling microscope, an image of a graphite surface immersed in deionized water was obtained with features less than 3 angstroms apart clearly resolved. Further, an image measured in saline solution demonstrated that the instrument can be operated under conditions useful for many biological samples.     

Micrometer- and nanometer-sized polymeric light-emitting diodes

A method for the fabrication of micrometer-and submicrometer-sized polymeric light-emitting diodes is presented. Such diodes have a variety of applications. Light sources of dimensions around 100 nanometers are required for subwavelength, near-field optical microscopy. Another possible application is patterning on the micrometer and nanometer scale. The diodes have been made in the form of a sandwich structure, with the conductive polymer poly(3,4-ethylene-dioxythiophene) polymerized in the pores of commercially available microfiltration membranes defining the hole-injecting contacts, poly[3-(4-octylphenyl)-2,2;-bithiophene] as the light-emitting layer, and a thin film of calcium-aluminum as the electron injector.     

Near-field microscopy through a SiC superlens

The wave nature of light limits the spatial resolution in classical microscopy to about half of the illumination wavelength. Recently, a new approach capable of achieving subwavelength spatial resolution, called superlensing, was invented, challenging the already established method of scanning near-field optical microscopy (SNOM). We combine the advantages of both techniques and demonstrate a novel imaging system where the objects no longer need to be in close proximity to a near-field probe, allowing for optical near-field microscopy of subsurface objects at sub-wavelength-scale lateral resolution.     

Video-rate far-field optical nanoscopy dissects synaptic vesicle movement

We present video-rate (28 frames per second) far-field optical imaging with a focal spot size of 62 nanometers in living cells. Fluorescently labeled synaptic vesicles inside the axons of cultured neurons were recorded with stimulated emission depletion (STED) microscopy in a 2.5-micrometer by 1.8-micrometer field of view. By reducing the cross-sectional area of the focal spot by about a factor of 18 below the diffraction limit (260 nanometers), STED allowed us to map and describe the vesicle mobility within the highly confined space of synaptic boutons. Although restricted within boutons, the vesicle movement was substantially faster in nonbouton areas, consistent with the observation that a sizable vesicle pool continuously transits through the axons. Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time.     

Subparticle ultrafast spectrum imaging in 4D electron microscopy

Single-particle imaging of structures has become a powerful methodology in nanoscience and molecular and cell biology. We report the development of subparticle imaging with space, time, and energy resolutions of nanometers, femtoseconds, and millielectron volts, respectively. By using scanning electron probes across optically excited nanoparticles and interfaces, we simultaneously constructed energy-time and space-time maps. Spectrum images were then obtained for the nanoscale dielectric fields, with the energy resolution set by the photon rather than the electron, as demonstrated here with two examples (silver nanoparticles and the metallic copper-vacuum interface). This development thus combines the high spatial resolution of electron microscopy with the high energy resolution of optical techniques and ultrafast temporal response, opening the door to various applications in elemental analysis as well as mapping of interfaces and plasmonics.     

Subnanometer-Diameter Wires Isolated in a Polymer Matrix by Fast Polymerization

The preparation and analysis of inorganic-organic polymer nanocomposites consisting of inorganic nanowires and multiwire "cables" in a random-coil organic polymer host is reported. Dissolution of inorganic (LiMo3Se3)n wires in a strongly coordinating monomer, vinylene carbonate, and the use of a rapid polymerization in the presence of a cross-linking agent produce nanocomposites without phase separation. Polymerization of dilute solutions yields a material containing mostly (Mo3Se3(-))n mono- and biwires, 6 to 20 angstroms in diameter and 50 to 100 nanometers long. Polymerization of more concentrated liquid crystalline solutions yields a nanocomposite containing oriented multiwire cables, 20 to 40 angstroms in diameter and up to 1500 nanometers long, that display optical anisotropy and electrical conductivity.     

Nanoelectromechanical systems

Nanoelectromechanical systems are evolving, with new scientific studies and technical applications emerging. Mechanical devices are shrinking in thickness and width to reduce mass, increase resonant frequency, and lower the force constants of these systems. Advances in the field include improvements in fabrication processes and new methods for actuating and detecting motion at the nanoscale. Lithographic approaches are capable of creating freestanding objects in silicon and other materials, with thickness and lateral dimensions down to about 20 nanometers. Similar processes can make channels or pores of comparable dimensions, approaching the molecular scale. This allows access to a new experimental regime and suggests new applications in sensing and molecular interactions.     

Preparation of photonic crystals made of air spheres in titania

Three-dimensional crystals of air spheres in titania (TiO2) with radii between 120 and 1000 nanometers were made by filling the voids in artificial opals by precipitation from a liquid-phase chemical reaction and subsequently removing the original opal material by calcination. These macroporous materials are a new class of photonic band gap crystals for the optical spectrum. Scanning electron microscopy, Raman spectroscopy, and optical microscopy confirm the quality of the samples, and optical reflectivity demonstrates that the crystals are strongly photonic and near that needed to exhibit band gap behavior.     

Atomic force microscopy and dissection of gap junctions

An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers.     

Optical projection tomography as a tool for 3D microscopy and gene expression studies

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.     

Photothermal imaging of nanometer-sized metal particles among scatterers

Ambient optical detection of labeled molecules is limited for fluorescent dyes by photobleaching and for semiconducting nanoparticles by "blinking" effects. Because nanometer-sized metal particles do not optically bleach, they may be useful optical labels if suitable detection signals can be found. We demonstrate far-field optical detection of gold colloids down to diameters of 2.5 nanometers with a photothermal method that combines high-frequency modulation and polarization interference contrast. The photothermal image is immune to the effects of scattering background, which limits particle imaging through Rayleigh scattering to diameters larger than 40 nanometers.     

Discovery of the atomic oxygen green line in the Venus night airglow

Green line emission at 557.7 nanometers arising from the O(1S - 1D) transition of atomic oxygen has been observed on the nightside of Venus with HIRES, the echelle spectrograph on the W. M. Keck I 10-meter telescope. We also observe optical emissions of molecular oxygen, consistent with the spectra from the Venera orbiters, but our green line intensity is so high that we cannot explain how it could be inconspicuous in the Venera spectra. An upper limit for the intensity of the O(1D - 3P) oxygen red line at 630 nanometers has also been obtained. The large green/red ratio indicates that the source is not associated with the Venus ionosphere. An important conclusion is that observation of the green line in a planetary atmosphere is not an indicator of an atmosphere rich in molecular oxygen.     

Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.     

High-resolution scanning x-ray diffraction microscopy

Coherent diffractive imaging (CDI) and scanning transmission x-ray microscopy (STXM) are two popular microscopy techniques that have evolved quite independently. CDI promises to reach resolutions below 10 nanometers, but the reconstruction procedures put stringent requirements on data quality and sample preparation. In contrast, STXM features straightforward data analysis, but its resolution is limited by the spot size on the specimen. We demonstrate a ptychographic imaging method that bridges the gap between CDI and STXM by measuring complete diffraction patterns at each point of a STXM scan. The high penetration power of x-rays in combination with the high spatial resolution will allow investigation of a wide range of complex mesoscopic life and material science specimens, such as embedded semiconductor devices or cellular networks.     

Molecular Nanotube Aggregates of beta- and ggr-Cyclodextrins Linked by Diphenylhexatrienes

Linked by strings of diphenylhexatriene (DPH) molecules, beta- and gamma-cyclodextrins (CDs) can form nanotube aggregates that contain as many as approximately 20 betaCDs (20 nanometers long) or approximately 20 to 35 gammaCDs (20 to 35 nanometers long). Nanotube formation was indicated in solution, by fluorescence anisotropy and light scattering results, and on graphite surfaces, by scanning tunneling microscopy. Tubes were not observed for the smaller alphaCDs. Molecular modeling shows that CD cavity size and the rodlike DPH structure are key factors in nanotube formation. Spectra generated by proton nuclear magnetic resonance indicate the inclusion of DPH in the interior of the CDs and formation of nanotubes in betaCDs and gammaCDs only. The photophysical properties of DPH are affected by its arrangement into a one-dimensional array within the CD nanotube, possibly because of exciton formation.