Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae: 2. Role of Filamentous Extracellular Network and Cell-to-Cell Connections at the Biofilm Surface

Flavobacterium johnsoniae forms a thin spreading colony on nutrient-poor agar using gliding motility. As reported in the first paper, WT cells in the colony were sparsely embedded in self-produced extracellular polymeric matrix (EPM), while sprB cells were densely packed in immature biofilm with less matrix. The colony surface is critical for antibiotic resistance and cell survival. We have now developed the Grid Stamp-Peel method whereby the colony surface is attached to a TEM grid for negative-staining microscopy. The images showed that the top of the spreading convex WT colonies was covered by EPM with few interspersed cells. Cells exposed near the colony edge made head-to-tail and/or side-to-side contact and sometimes connected via thin filaments. Nonspreading sprB and gldG and gldK colonies had a more uniform upper surface covered by different EPMs including vesicles and filaments. The EPM of sprB, gldG, and WT colonies contained filaments ~2 nm and ~5 nm in diameter; gldK colonies did not include the latter. Every cell near the edge of WT colonies had one or two dark spots, while cells inside WT colonies and cells in SprB-, GldG-, or GldK-deficient colonies did not. Together, our results suggest that the colony surface structure depends on the capability to expand biofilm.

Quartz Crystal Microbalance Genosensing of Brettanomyces Bruxellensis Yeast in Wine Using a Rapid and Efficient Drop and Collect Protocol

A miniaturized quartz crystal microbalance (QCM) genosensor is proposed for sensitive and real-time detection of short ssDNA sequences (53 bp) or DNA extracted from Brettanomyces bruxellensis (Brett) yeast cells. The presence of Brett yeast causes a depreciation of the quality of aged fine wines, producing molecules of unpleasant odors and biogenic amines that are harmful to human health. More specifically, standard quartz crystal (S-QCM) and homemade 4 nm gold transmission electron microscopy (TEM)-grid patterned quartz (multi-TEM QCM) are herein proposed for biofunctionalization steps with different ssDNA sequences. By employing a rapid and efficient drop and collect protocol, the specific detection of 1 pg/µL ssDNA Brett of a short sequence and 100 ng/μL DNA of B. bruxellensis extracted from a wine sample (VR2008) is reported.

Local Substrate Heterogeneity Influences Electrochemical Activity of TEM Grid-Supported Battery Particles

Understanding how particle size and morphology influence ion insertion dynamics is critical for a wide range of electrochemical applications including energy storage and electrochromic smart windows. One strategy to reveal such structure–property relationships is to perform ex situ transmission electron microscopy (TEM) of nanoparticles that have been cycled on TEM grid electrodes. One drawback of this approach is that images of some particles are correlated with the electrochemical response of the entire TEM grid electrode. The lack of one-to-one electrochemical-to-structural information complicates interpretation of genuine structure/property relationships. Developing high-throughput ex situ single particle-level analytical techniques that effectively link electrochemical behavior with structural properties could accelerate the discovery of critical structure-property relationships. Here, using Li-ion insertion in WO3 nanorods as a model system, we demonstrate a correlated optically-detected electrochemistry and TEM technique that measures electrochemical behavior of via many particles simultaneously without having to make electrical contacts to single particles on the TEM grid. This correlated optical-TEM approach can link particle structure with electrochemical behavior at the single particle-level. Our measurements revealed significant electrochemical activity heterogeneity among particles. Single particle activity correlated with distinct local mechanical or electrical properties of the amorphous carbon film of the TEM grid, leading to active and inactive particles. The results are significant for correlated electrochemical/TEM imaging studies that aim to reveal structure-property relationships using single particle-level imaging and ensemble-level electrochemistry.

A Solution for Obtaining an Advanced Lamella Geometry on the Grid with a Single Manipulation Step

A protocol for obtaining an advanced TEM lamella geometry using FIB-SEM is presented. Lamella lift-out procedure might require multiple manipulation steps or even breaking the vacuum in order to reach inverted or plan-view lamella geometries. We have developed a setup which enables lamella transfer from a bulk sample onto a TEM grid within a single, very simple manipulation step, with no need to break the vacuum or unload the sample. Most importantly, this approach does not require any additional devices to be installed.

Sublimable materials facilitate the TEM sample preparation of oil-soluble nanomaterials

Sample preparation is significantly important to the high-resolution transmission electron microscopy (HRTEM) characterization of nanomaterials. However, many general organic solvents can dissolve the necessary organic polymer support layer in TEM grid, which causes it difficult to obtain high-quality samples of oil-soluble nanomaterials. In this study, a new sample preparation method for oil-soluble nanomaterials has been developed by using the sublimable material as a transition layer. Experiments also show that there is no damage to TEM grids and high-quality HRTEM images can be obtained via this method. This approach paves the way to applicable HRTEM sample preparation of oil-soluble nanomaterials.