Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution.

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.

Three-dimensional chemical mapping by scanning transmission X-ray spectromicroscopy.

Three-dimensional (3-d) chemical mapping using angle-scan tomography in a scanning transmission X-ray microscope is demonstrated. Apparatus, experimental procedures and data processing are presented and the 3-d spatial resolution is evaluated. The technique is illustrated using mapping of a low-density acrylate polyelectrolyte in and outside of polystyrene microspheres dispersed in water in a 4 microm-diameter microcapillary. The 3-d chemical visualization provides information about the microstructure that had not previously been observed.

Interface quality and thermal stability of laser-deposited metal/MgO multilayers.

Metal/MgO multilayers (metal of Fe, Ni80Nb20, and Ti) with bilayer periods in the range 1.2-3.0 nm have been prepared by pulsed laser deposition and characterized by both hard and soft-x-ray reflectometry. The interface roughness is found to be < or = 0.5 nm in all the samples and is nearly independent of the total number of deposited bilayers. The interface roughness, however, depends on the absolute thickness of the individual layers and increases from approximately 0.3 nm for a 3.0-nm period to approximately 0.5 nm for a bilayer period of 1.2 nm. The multilayers are found to be highly stable up to temperatures as high as 550 degrees C. The hard-x-ray reflectivity of the multilayers decreases for T > 300 degrees C, whereas the layered structure is stable up to 550 degrees C. The reflectivity in the water window region of soft x rays, lambda = 3.374 nm, was found to be 0.4% at an angle of incidence of approximately 54 degrees for multilayers with 60 bilayers at a period of approximately 2.1 nm.

Exploring the use of soft X-ray microscopy for imaging subcellular structures of the inner ear.

The soft X-ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X-ray microscopy provides high-resolution visualization of hydrated, non-embedded and non-sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X-ray micrographs of structures isolated from the guinea-pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X-ray images of the acellular tectorial membrane and thin two-layered Reissner's membrane display a level of resolution comparable to low-power electron microscopy.

14.5% near-normal incidence reflectance of Cr/Sc x-ray multilayer mirrors for the water window.

Cr/Sc multilayer mirrors, synthesized by ion-assisted magnetron sputter deposition, are proved to have a high near-normal reflectivity of R = 14.5% at a grazing angle of 87.5 degrees measured at the wavelength lambda = 3.11 nm, which is an improvement of more than 31% compared with previously published results. Elastic recoil detection analyses show that the mirrors contained as much as 15 at. % of N and traces of C and O. Soft x-ray reflectivity simulations reveal interface widths of sigma = 0.34 nm and an exceptionally small layer thickness drift of approximately 1.6 x 10(-5) nm/multilayer period throughout the stack. Simulations show that a reflectivity of R = 25.6% is attainable if impurities and layer thickness drift can be eliminated. The abrupt interfaces are achieved with ion assistance with a low ion energy of 24 eV and high ion-to-metal flux ratios of 7.1 and 23.1 during Cr and Sc sputter deposition, respectively. In addition, a near-normal incidence reflectivity of 5.5% for the C VI emission line (lambda = 3.374 nm) from a laser plasma source was verified.