Tseng-Ming Chou

Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-alpha by macrophages.

Despite the near universal association of congenital heart block and maternal Abs to SSA/Ro and SSB/La, the intracellular location of these Ags has made it difficult to substantiate their involvement in pathogenicity. To define whether components of the SSA/Ro-SSB/La complex, which translocate during apoptosis, are indeed accessible to extracellular Abs, two approaches were taken: immunoprecipitation of surface biotinylated proteins and scanning electron microscopy. Human fetal cardiocytes from 16–24-wk abortuses were cultured and incubated with staurosporine to induce apoptosis. Surface biotinylated 48-kDa SSB/La was reproducibly immunoprecipitated from apoptotic, but not nonapoptotic cardiocytes. Surface expression of SSA/Ro and SSB/La was further substantiated by scanning electron microscopy. Gold particles (following incubation with gold-labeled sera containing various specificities of anti-SSA/Ro-SSB/La Abs and murine mAb to SSB/La and 60-kDa SSA/Ro) were consistently observed on early and late apoptotic cardiocytes. No particles were seen after incubation with control antisera. To evaluate whether opsonized apoptotic cardiocytes promote inflammation, cells were cocultured with macrophages. Compared with nonapoptotic cardiocytes or apoptotic cardiocytes incubated with normal sera, apoptotic cardiocytes preincubated with affinity-purified Abs to SSB/La, 52-kDa SSA/Ro, or 60-kDa SSA/Ro increased the secretion of TNF-α from cocultured macrophages. In summary, apoptosis results in surface accessibility of all SSA/Ro-SSB/La Ags for recognition by circulating maternal Abs. It is speculated that in vivo such opsonized apoptotic cardiocytes promote an inflammatory response by resident macrophages with damage to surrounding conducting tissue.

Transmission electron holography of silicon nanospheres with surface oxide layers

Phase images of 20–30-nm-diam silicon spheres were collected by holographic methods in a field-emissiontransmission electron microscope. The spherical geometry enables the effect of specimen thickness on the electron-wave phase to be separated from the intrinsic Si electron-optical refractive effects allowing a determination of the mean inner potential Φ 0 . This work finds Φ 0 =11.9±0.9 V characterizing amorphous Si and 12.1±1.3 V characterizing crystalline Si. The phase images can resolve a 2-nm-thick native oxide layer and give Φ 0 for SiO 2 =10.1±0.6 V. The phase data can quickly recognize a surface layer, and the effect of a surface layer on the determination of the bulk mean potential can be minimized.

Extracellular matrix reorganization during cryo preparation for scanning electron microscope imaging of Staphylococcus aureus biofilms.

Biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM). They can be visualized by scanning electron microscopy (SEM), but the requisite SEM sample preparation can modify the biofilm morphology. Here, four different approaches to prepare biofilms of hydrated Staphylococcus aureus for SEM imaging are compared. In order of increasing cooling effectiveness these are: (1) drying in air; (2) plunging in liquid nitrogen; (3) plunging in liquid ethane; and (4) high pressure freezing with liquid nitrogen. These different methods give rise to markedly different biofilm morphologies, which are revealed by cryo-SEM imaging. Significantly, high-pressure frozen biofilms exhibit a rich network of nanoscale ECM fibers surrounding individual bacteria throughout the biofilm thickness. This structure is entirely lost when similar biofilms are dried in air, and it is substantially modified when these biofilms are plunged into liquid nitrogen or liquid ethane.

Mean free paths for inelastic electron scattering in silicon and poly(styrene) nanospheres

The mean free paths for inelastic electron scattering, lambda(in), in silicon [Si] and poly(styrene) [PS] have been measured using off-axis electron holography in a field-emission transmission electron microscope (FEG TEM). The holographic imaging method determines both quantitative wave phase information as well as elastic energy-filtered wave amplitude information. Using the energy-filtered amplitude data, two-dimensional t/lambda(in) images are reconstructed. The present work uses spherical nanoparticles as samples, so the sample thickness at any point in a two-dimensional image can be calculated knowing the center and radius of the projected nanosphere. The thickness contribution to t/lambda(in) is removed to obtain quantitative lambda(in) values. This work finds values of lambda(i)Si = 53.8 +/- 5.5 and 88.6 +/- 6.9 nm, and lambda(PS) = 78.1 +/- 3.4 and 113.0 +/- 5.9 nm for 120 and 200 keV incident electron energies, respectively.

Quantitative phase contrast imaging of arborescent graft polystyrene by off-axis transmission electron holography

Imaging nanoscale polymer objects in the Transmission Electron Microscope is difficult, because small polymeric objects interact only weakly with intermediate-energy electrons. Heavy element staining can induce significant amplitude contrast, but stains can introduce artifacts that complicate the structure determination at nanometer length scales. This paper explores transmission electron holography for phase contrast imaging of unstained arborescent graft polystyrene nanoparticles. Holography is able to recover significant phase contrast from these particles despite the fact that there is negligible amplitude contrast. Comparative imaging experiments show that off-axis holography provides substantially higher contrast than that generated by the traditional method of transferring phase information to amplitude information via defocus. This effect is a consequence of different lens contrast-transfer behavior in each of these two imaging approaches. Under kinematical conditions when the appropriate mean inner potential is known, the specimens projected thickness can be directly mapped from the holographic phase image to give a measure of the specimens three-dimensional shape. Such quantitative imaging shows that individual arborescent graft polystyrene nanoparticles, which are spherical in a good solvent, adopt a flattened shape when deposited on a carbon substrate and allowed to dry.

Reorganization of S. aureus ECM during Cryo-Preparation for SEM Imaging

Biofilms are 3-D communities of bacteria distributed in a highly hydrated extracellular matrix (ECM) [1]. ECM formation and structuring is an important hallmark characteristic of a mature biofilm. Many biofilms, including those of staphylococci, produce ECM that contains polysaccharides, proteins, and extracellular DNA. These substances are all highly hydrophilic, and the water content of a biofilm can be as high as 90%. Here we study 4 methods to prepare hydrated biofilms of Staphylococcus aureus (S. aureus) for (cryo-)SEM imaging [2]. These different preparation methods give rise to markedly different biofilm morphologies as revealed by cryo-SEM imaging at different stages during ice sublimation. The specimens were all imaged using low-keV electrons in a Zeiss Auriga dual-beam FIB-SEM.

Cryo-SEM Imaging and Analysis of Frozen-Hydrated PEG-AA Microgels

Plunge freezing is a common method to prepare hydrated samples for cryo-SEM. Thin specimens are rapidly immersed in liquid nitrogen and cryo-transferred to the cryo-SEM. Fig. 1a shows an example from a cryo-plunged aqueous colloidal solution of PEG-AA microgels. While a hydrated microgel should have a smooth surface due to surface-tension effects, fig. 1a shows microgels with rough surfaces. Roughness is presumably due to microgel reorganization during water crystallization. In contrast, Fig. 1b shows an identical specimen prepared by high-pressure freezing (Leica HPM-100 HPF). The HPF method gives rise to frozen-hydrated microgels each having a very smooth surface. HPF is more effective in freezing water to its amorphous state and thus preserves the intrinsic microgel morphology. Similar effects occur during the cryo-preparation of thick bacterial biofilms [4].

FIB Tomography of Bacterial Biofilms Grown on Gold and Polystyrene

Bacterial biofilms are three-dimensional communities of bacteria distributed in a highly hydrated extracellular matrix (ECM) (Fig.1). They have structures over multiple length scales ranging from nanoscale to macroscale. Following standard EM sample-preparation protocols of embedding cellular samples [1], we can fix, stain, and embed biofilms while maintaining the close to their native state. This enables us to study their three-dimensional structure using focused ion beam (FIB) tomography [2].

Temporospatial Relationship of Lipid Droplets and Mitochondria in Cardiac Muscle by Focused Ion Beam Scanning Electron Microscopy

Obesity is a rising health problem in the United States and many developed countries. The World Health Organization estimates that, in 2014, more than 1.9 billion adults were overweight worldwide, and more than 600 millions were obese [1]. Obesity is the result of an imbalance in lipid homeostasis and is evidenced by the excessive accumulation of cytoplasmic lipid droplets (CLDs) in cardiac muscles. The CLD is a dynamic organelle that interacts with various cell organelles such as the endoplasmic reticulum, endosomes and mitochondria. Our previous studies suggested that the LD specific protein, perilipin 5, is involved in promoting association of mitochondria to LDs [2]. Here we investigate the temporospatial relationship of LDs, mitochondria, endoplasmic reticulum (sarcoplasmic reticulum) and muscle fibrils in a 3D volume of mouse cardiac muscle using a focused ion beam scanning electron microscope (FB-SEM).

Cryo-FIB Minimizes Ga + Milling Artifacts in Sn

Since FIB was first introduced, one of the major applications has been to mill site-specific crosssectioned sample surfaces [1]. This approach not only allows SEM access to the structural information buried under the surface, but it is also the first step of making TEM lamellae using FIB. Importantly, there are many reports of beam-induced damage due to FIB processing. Here, we report on artifacts induced when milling metallic Sn with Ga ions and how this problem can be minimized via cryo-FIB.