Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formation in vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to the in vivo pattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it models in vivo organization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

All common negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-Å (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide negative staining of single particle specimens. Appositional contrast is less than that given by heavy metal negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 Å. Future experimental use of sodium phosphate buffer and magnesium sulfate for negative staining is anticipated, particularly in designing new cocktail (multicomponent) negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.

Biofilms are assemblages of microorganisms and their associated extracellular products at an interface and typically with an abiotic or biotic surface. The study of the morphology of biofilms is important because they are associated with processes of biofouling, corrosion, catalysis, pollutant transformation, dental caries, drug resistance, and so forth. In the literature, biofilms have been examined by atomic force microscopy (AFM), which has proven to be a potent tool to study different aspects of the biofilm development on solid surfaces. In this work, we used AFM to investigate topographical changes during the development process of Enterococcus faecalis biofilms, which were generated on sterile cellulose nitrate membrane (CNM) filters in brain heart infusion (BHI) broth agar blood plates after 24, 36, 72, 192, and 360 h. AFM height images showed topographical changes due to biofilm development, which were used to characterize several aspects of the bacterial surface, such as the presence of extracellular polymeric substance, and the biofilm development stage. Changes in the development stage of the biofilm were shown to correlate with changes in the surface roughness as quantified through the mean roughness.

Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.

An energy dispersive X-ray spectrometry (EDXS) method is developed to evaluate the composition of alloyed nanoparticles (NPs) where one of the alloying elements is removed under the electron beam during microanalysis with a transmission electron microscope (TEM). The method is demonstrated for alloyed Au-Ag NPs of a diameter ranging from 6 to 20 nm produced by laser evaporation of a water-suspended Ag-Au powder mixture of varying composition. Series of EDXS spectra are recorded for 30 NPs from samples with five different Ag:Au ratios revealing Ag depletion from NPs during electron irradiation. By studying the evolution of NPs composition as a function of dose, the initial Ag content for each NP is extrapolated. The rate of Ag depletion is discussed in terms of sputtering and knock-on damage. On average, approximately one Ag atom is lost from the NP for each Ag L X-ray detected. To assess the limitations of microanalysis in these sensitive nanoscale structures, the concept of detectability limit is adapted to our method. This benchmark is then evaluated for Ag in Au-Ag NPs of various sizes and acquisition times. This study should be regarded as a guide for the design of analytical TEM measurements of beam-sensitive NPs.

The development of a remotely operated scanning electron microscopy (SEM) system and its use by high school students and the public as an outreach program are reported. The SEM and the server are located in the National Institute for Materials Science, Tsukuba, Japan, with client computers installed at a science museum and high schools. Using a secure virtual private network system and scheduling/management groupware, observation of SEM images and energy dispersive X-ray analysis are widely and frequently performed throughout Japan.

A luminescence database for minerals and materials has been complied from the literature, the aim being to create a resource that will aid in the analysis of luminescence spectral of ionic species in minerals and materials. The database is based on a range of excitation techniques and records both major and minor lines, and their activators. The luminescence techniques included in the database are cathodoluminescence, ion luminescence, and photoluminescence. When combined with other traditional X-ray measurements collected on the same region, use of the luminescence database will give additional insight into the chemistry of minerals and materials.

Force Microscopy: Applications in Biology and Medicine. Edited by Bhanu P. Jena and J.K. Heinrich Hörber. John Wiley & Sons, Inc., Hoboken, NJ; 2006, 300 pages. ISBN 978-0-471-39628-4

The excitement of man's imagination and discovery by the scope of imaging tools, whether tele- or micro-, from 1015 to 10−15 m, ranges from the limits of the known universe to the atomic level. Light and electron microscopy opened vistas of biology and medicine with discovery of the unit of life, the cell and viruses. In the mid-1980s development of scanning probe microscopy allowed studies on live cells at the near-nanometer level. Thus, results from studies using the atomic force microscope (AFM) have given birth to a new field, “NanoCellBiology”, by providing investigators a three-dimensional view of the structure and dynamics of live cells and biomolecules at near-angstrom resolution. Applications of force microscopy to the study of physiological processes of living cells combined with biochemical and molecular biology are yielding rapid advances on how normal and diseased cells function. This exciting book contains 15 chapters by invited experts that examine cell secretory machinery and the discovery of a new cellular structure—the porosome—involved in cell secretion, properties of microbial and cell plant surfaces, cellular interactions of nanodrug delivery systems, intermolecular forces and avidity modulation of leukocyte adhesion molecules, micromechanical properties of lipid bilayers and vesicles, imaging of soft surfaces, high-speed action of biomolecules in motion, cytogenetic applications, and the study of macromolecular interactions in hemostasis and thrombosis.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at http://www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to Nan Yao, nyao@Princeton.edu.