One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) (air plant, miracle leaf) is popularly used to treat gastrointestinal disorders and wounds. Recently, the species was tested to treat cutaneous leishmaniasis with successful results. This medicinal activity was associated with the phenolic fraction of the plant. Blue light induces biosynthesis of phenolic compounds and many changes in anatomical characteristics. We studied the effects of supplementary blue light on the leaf morphology of in vitro K. pinnata. Plants cultured under white light (W plants) only and white light plus blue light (WB plants) show petioles with plain-convex section, amphistomatic leaf blades with simple epidermis, homogeneous mesophyll with densely packed cells, and a single collateral vascular bundle in the midrib. W plants have longer branches, a larger number of nodes per branch, and smaller leaves, whereas WB plant leaves have a thicker upper epidermis and mesophyll. Leaf fresh weight and leaf dry weight were similar in both treatments. Phenolic idioblasts were observed in the plants supplemented with blue light, suggesting that blue light plays an important role in the biosynthesis of phenolic compounds in K. pinnata.

A simple fluorescence microscopy technique is developed and presented to investigate heterogeneities in emission intensity and quenching responses of luminescence sensors and to measure diffusion and permeation coefficients of oxygen in polymers. Most luminescence oxygen sensors do not follow linearity of the Stern-Volmer (SV) equation due to heterogeneity of luminophore in the polymer matrix. To circumvent this limitation, inverted fluorescence microscopy is utilized in this work to investigate the SV response of the sensors at the micron scale. It was found that intensity is higher in regions where the luminophore is aggregated, but the response is poorer to oxygen concentration. In contrast, the nearly homogeneous regions exhibit linearity with high SV constants. In these diffusion experiments, oxygen concentration was measured by luminescence changes in regions with high SV constants and good linearity. Two diffusion experiments were performed—termed film-on-sensor and accumulation-in-volume techniques. A new Fick's law based quasi-steady-state diffusion model was developed and combined with the SV equation to obtain effective permeation coefficients for the accumulation-in-volume technique. Using these experimental techniques, oxygen diffusion properties in free-standing Teflon polymer films, cast silicon elastomers, and cast polydimethylsiloxane films containing different weight percentages of zeolite were determined with good precision.

Electron holography has been shown to allow a posteriori aberration correction. Therefore, an aberration corrector in the transmission electron microscope does not seem to be needed with electron holography to achieve atomic lateral resolution. However, to reach a signal resolution sufficient for detecting single light atoms and very small interatomic fields, the aberration corrector has turned out to be very helpful. The basic reason is the optimized use of the limited number of “coherent” electrons that are provided by the electron source, as described by the brightness. Finally, quantitative interpretation of atomic structures benefits from the holographic facilities of fine-tuning of the aberration coefficients a posteriori and from evaluating both amplitude and phase.

Effects related with the attenuation and deflection suffered by an electron beam when it passes through a carbon conductive coating and an oxide film layer on the surface of bulk samples are studied by Monte Carlo simulations and energy dispersive spectroscopy with electron excitation. Analytical expressions are provided for the primary beam energy and intensity losses and for the deflection of the incident electrons in both layers, in terms of the incidence energy, the film mass thicknesses, and the atomic number of the oxidized element. From these analytical expressions, suitable corrections are proposed for the models used to describe the X-ray spectrum of the substrate, including also the contribution of the X-rays generated in the oxide and conductive films and the characteristic X-ray absorption occurring in those layers. The corrections are implemented in a software program for spectral analysis based on a routine of parameter refinement, and their influence is studied separately in experimental spectra of single-element standards measured at different excitation energies. Estimates for the layer thicknesses are also obtained from the spectral fitting procedure.

Deposition of calcium carbonate from groundwater was examined on a moss, Didymodon tophaceus, from a Niagara Escarpment waterfall. A spectrophotometer on a polarizing microscope was used for interferometry. A second-order blue interference with an interference minimum around 620 nm was found when moss cell spaces were fully calcified. Filled cell spaces were often surrounded by empty cell spaces. Complete calcification of whole leaflets resulted in progressively higher orders of interference colors and a positive shift in interference minima. Calcified leaflets finally became cemented together, but each retained a weak extinction when rotated. Small calcareous spherulites (mean diameter 15.7 ± 2.1 μm) were found between leaflets. Spherulites exhibited first-order white interference with a Maltese cross that rotated when the polarizer and analyzer were rotated in tandem. A Nikitin-Berek compensator was tilted at 5.5° to give an interference minimum at 600 nm in the optical axis. Quadrants of spherulites with radii more or less in line with the tilting axis of the compensator had lower (P < 0.001) interference minima (535 ± 27 nm) than quadrants with radii more or less perpendicular to the compensator (659 ± 15 nm), thus indicating a radial internal structure. Spherulites were tentatively identified as vaterite.

Neurodegerative disorders affect millions of people worldwide. Neural cells derived from human embryonic stem cells (hESC) have the potential for cell therapies and/or compound screening for treating affected individuals. While both protein and gene expression indicative of a neural phenotype has been exhibited in these differentiated cells, ultrastuctural studies thus far have been lacking. The objective of this study was to correlate hESC to neural differentiation culture conditions with ultrastructural changes observed in the treated cells. We demonstrate here that in basic culture conditions without growth factors or serum we obtain neural morphology. The addition of brain-derived neurotrophic factor (BDNF) and serum to cultures resulted in more robust neural differentiation. In addition to providing cues such as cell survival or lineage specification, additional factors also altered the intracellular structures and cell morphologies. Even though the addition of BDNF and serum did not increase synaptic formation, altered cellular structures such as abundant polyribosomes and more developed endoplasmic reticulum indicate a potential increase in protein production.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Graphene is a single layer of carbon atoms arranged in a hexagonal lattice. The high carrier mobility and mechanical robustness of single layer graphene make it an attractive material for “beyond CMOS” devices. The current work investigates through high-resolution transmission electron microscopy (HRTEM) image simulation the sensitivity of aberration-corrected HRTEM to the different graphene stacking configurations AAA/ABA/ABC as well as bilayers with rotational misorientations between the individual layers. High-angle annular dark field–scanning transmission electron microscopy simulation is also explored. Images calculated using the multislice approximation show discernable differences between the stacking sequences when simulated with realistic operating parameters in the presence of low random noise.

Magnetic nanocomposite materials consisting of 5 and 10 wt% CoFe2O4 nanoparticles in a silica aerogel matrix have been synthesized by the sol-gel method. For the CoFe2O4-10wt% sample, bright-field scanning transmission electron microscopy (BF STEM) and high-resolution transmission electron microscopy (HREM) images showed distinct, rounded CoFe2O4 nanoparticles, with typical diameters of roughly 8 nm. For the CoFe2O4-5wt% sample, BF STEM images and energy dispersive X-ray (EDX) measurements showed CoFe2O4 nanoparticles with diameters of roughly 3 ± 1 nm. EDX measurements indicate that all nanoparticles consist of stoichiometric CoFe2O4, and electron energy-loss spectroscopy measurements from lines crossing nanoparticles in the CoFe2O4-10wt% sample show a uniform composition within nanoparticles, with a precision of at best than ±0.5 nm in analysis position. BF STEM images obtained for the CoFe2O4-10wt% sample showed many “needle-like” nanostructures that typically have a length of ∼10 nm and a width of ∼1 nm, and frequently appear to be attached to nanoparticles. These needle-like nanostructures are observed to contain layers with interlayer spacing 0.33 ± 0.1 nm, which could be consistent with Co silicate hydroxide, a known precursor phase in these nanocomposite materials.

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or ▵1-90+▵267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

The organic interprismatic layers of the mollusc Pinctada margaritifera are studied using a variety of highly spatially-resolved techniques to establish their composition and structure. Our results show that both the interlamellar sheets of the nacre and interprismatic envelopes form layered structures. Additionally, these organic layers are neither homogeneous in composition, nor continuous in their structure. Both structures play a major role in the biomineralization process and act as a boundary between mineral units.

A lens system is proposed that not only provides spherical correction of the objective lens by charges that are induced on a thin foil, in the way proposed in a paper by Otto Scherzer [Optik56(2), 133–147, 1980], but also provides Zernike phase contrast by means of an appropriate phase shift of the scattered electrons within the foil. This system has the potential to provide strong phase contrast from very low spatial frequencies to frequencies above 1/(100 pm).

The discoloring interaction between the artist's pigments cadmium yellow and the copper-containing malachite, an interaction that is conjectured to cause black spotting in oil paintings of the 19th and early 20th centuries, was examined using X-ray mapping and scatter diagram analysis. The application of these coupled techniques confirmed that copper sulfide phases were produced during discoloration reaction. Scatter diagram analysis indicated that two copper sulfide stoichiometries (CuS and Cu3S2) were present as reaction products where previously only crystalline CuS (covellite) had been identified by X-ray diffraction. The results demonstrate the potential of X-ray mapping coupled with scatter diagram analysis for the identification of both crystalline and X-ray amorphous phases produced by such complex heterogeneous interactions and their applicability to the investigation of interactions of artists' pigments.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.