Improving photon counts for STORM with COT.

<p>(<b>a</b>) Mean photon counts measured for three different STORM buffers using different thiols (MEA and/or BME) and the same oxygen scavenging system (glucose oxidase/catalase, <b>see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a> &</b> <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#s3" target="_blank">Methods</a></b> for details on composition); number indicates the corresponding fold-increase upon addition of 2 mM COT (red bars) compared to no COT added (black bars) (pH ∼7.5) (<b>b</b>) Normalized photon count distributions for the “MEA+BME” buffer as a function of COT concentration (pH 8). (<b>c</b>) Mean photon count as a function of COT concentration for the buffer “MEA+BME” (pH 8).</p

Buffer-Enhanced 2D STORM imaging of microtubules.

<p>(<b>a</b>) Widefield, and (<b>b</b>) STORM image of a COS-7 cell stained with alpha-tubulin primary and Alexa-647-F(ab’)2 secondary antibodies, imaged in Buffer #4 (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>). (<b>c</b>) Zoom on the ROI (red box) defined in (b), number of localized molecules = 1960. (<b>d</b>) Lateral profiles taken either from a 200 nm-wide region (violet box) and corresponding curve shown in violet, or averaged over seven 200 nm-wide regions (inside the dashed red box) highlighted in (c) and the corresponding curve in red. (<b>e</b>) Averaged lateral profile shown in (d) fitted with a double Gaussian. (<b>f</b>) Model of the stained microtubule (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s004" target="_blank">Figure S4</a></b>). The labeled antibodies are expected to form a ring around the microtubule with an inner diameter of ∼25 nm and an outer diameter of ∼50 nm. (<b>g</b>) STORM image of COS7 cells strained with Cep152 primary and Alexa-647 F(ab’)2 secondary antibodies, imaged in Buffer #4 (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>). The top panel shows two tori from a side view, the bottom panel one torus in side views and one torus in cross-section. Scale bar is 1000 nm for (<b>a–c</b>) and 500 nm for (<b>g</b>).</p

Influence of pH on STORM imaging.

<p>(<b>a</b>) Decrease in pH as a function of time for the “BME+MEA” buffer containing glucose oxidase/catalase as oxygen scavenging system, (buffer #3 in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>) both with and without the addition of 2 mM COT. (<b>b</b>) Average photon counts per molecule as a function of pH for the “BME+MEA” buffer both with and without the addition of 2 mM COT. (<b>c</b>) pH as a function of time using the PCA/PCD oxygen scavenging system both with and without the addition of 2 mM COT (<b>d</b>) Normalized photon count distribution for the PCA/PCD buffer with and without COT at pH = 8 (mean photon count is 8,700 without COT and 32,000 with 2 mM COT).</p

Buffer-Enhanced 3D STORM imaging of Microtubules.

<p>COS-7 cells were stained with alpha-tubulin primary antibodies and Alexa-647 F(ab’)2 secondary antibodies and imaged. 3D-STORM images are color coded by depth.(<b>a–c</b>) Astigmatic 3D-STORM with an oil objective and PBS-Glycerol buffer (Buffer #5, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>)<b>:</b> (<b>a</b>) 3D-STORM image and corresponding axial profiles from 300×300 nm-wide regions taken (<b>b</b>) on the edge of the cell (dashed box) or (<b>c</b>) in a denser central region with microtubules crossings (full box).(<b>d-f</b>) Astigmatic 3D-STORM with a water objective and an index-matched buffer (Buffer #4, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>)<b>:</b> (<b>d</b>) 3D-STORM image and corresponding axial profiles from the 300×300 nm-wide regions taken (<b>e</b>) on the edge of the cell (dashed box) or (<b>f</b>) in a denser central region with microtubules crossings (full box). For each axial profile, positions of the fitted Gaussian peak maxima (green) as well as FWHM (blue) are indicated. Scale bar is 5 µm.</p

Buffer-Enhanced 3D STORM imaging on a biplane astigmatic microscope.

<p>(<b>a</b>) Schematics of the SR-200 inverted microscope (Vutara, Salt Lake City, UT). D: Dichroic mirror, F: Fluorescence filter, T: Tube lens, B: 50/50 Beamsplitter. Fluorescent light from a single molecule is collected by the objective and is imaged onto two different planes located on distinct parts of the EMCCD camera. The distance between the two planes is set by the optical path difference, and results in a measured axial shift of 780 nm. Optical aberrations are represented by in the schematics by the lateral shift in the position of the tube lens. (<b>b</b>) Measured PSF in the two planes which are used for 3D localization, shown at three different depths. (<b>c</b>) Biplane-Astigmatic 3D STORM image of a cell stained with Alexa-647-conjugated alpha-tubulin antibodies, color-coded by depth. Buffer #4 is used (See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069004#pone.0069004.s009" target="_blank">Table S1</a></b>) (<b>d,e</b>) Lateral and axial profiles were measured and averaged from five 200-nm wide regions. The blue line corresponds to a Gaussian fit, and the corresponding FWHM value is indicated. (<b>f</b>) Estimation of the resolution performed by convolving the projection of the known structure with a 40 nm wide Gaussian function, and the resulting expected distribution. Scale bar is 1 µm for (<b>b</b>) and 2.5 µm for (<b>c</b>).</p

Formation of Au Nanoparticles in Liquid Cell Transmission Electron Microscopy: From a Systematic Study to Engineered Nanostructures

In this work, a systematic study of the effect of electron dose rate, solute concentration, imaging mode (broad beam vs scanning probe mode), and liquid cell setup (static vs flow mode) on the growth mechanism and the ultimate morphology of Au nanoparticles (NPs) was performed in chloroauric acid (HAuCl₄) aqueous solutions using in situ liquid-cell TEM (LC-TEM). It was found that a diffusion limited growth dominates at high dose rates, especially for the solution with the lowest concentration (1 mM), resulting in formation of dendritic NPs. Growth of 2D Au plates driven by a reaction limited mechanism was only observed at low dose rates for the 1 mM solution. For the 5 mM and 20 mM solutions, reaction limited growth can still be induced at higher dose rates, due to abundance of the precursor available in the solutions, leading to formation of 2D plates or 3D faceted NPs. As a proof-of-concept, an Au nanostructure with a 3D faceted particle core and a dendritic shell can be in situ produced by simply tuning the electron dose in the 1 mM solution irradiated in a flow cell setup in the STEM mode. This work paves the way to study the growth of complex heteronanostructures composed of multiple elements in LC-TEM

Enhanced Carrier Collection from CdS Passivated Grains in Solution-Processed Cu₂ZnSn(S,Se)₄ Solar Cells

Solution processing of Cu₂ZnSn­(S,Se)₄ (CZTSSe)–kesterite solar cells is attractive because of easy manufacturing using readily available metal salts. The solution-processed CZTSSe absorbers, however, often suffer from poor morphology with a bilayer structure, exhibiting a dense top crust and a porous bottom layer, albeit yielding efficiencies of over 10%. To understand whether the cell performance is limited by this porous layer, a systematic compositional study using (scanning) transmission electron microscopy ((S)­TEM) and energy-dispersive X-ray spectroscopy of the dimethyl sulfoxide processed CZTSSe absorbers is presented. TEM investigation revealed a thin layer of CdS that is formed around the small CZTSSe grains in the porous bottom layer during the chemical bath deposition step. This CdS passivation is found to be beneficial for the cell performance as it increases the carrier collection and facilitates the electron transport. Electron-beam-induced current measurements reveal an enhanced carrier collection for this buried region as compared to reference cells with evaporated CdS