Whole-mount immunofluorescence technique provides a way to reveal integrated expression patterns of biological molecules in individuals. Well-documented morphological preservation ability in biology makes aldehydes the fixative of choice. Cross-linking among biocomponents and aldehydes is the key for maintaining morphology but masks the biological molecules for immunodetection. This study performs an easily accessible method by applying heat-induced retrieval, which can rescue the antigenicity of the proteins and also enhance the labeling sensitivity of the fluorescence dye in overfixed zebrafish embryos. The results show that the immunoreactivities of antibodies to myosin in the muscles, green fluorescent protein in the blood vessels and the nuclei in the cells can be recovered significantly, and the morphology of the zebrafish embryos, even the fragile mutants, is at the same time well maintained. Therefore, we provide a choice for antigen retrieval, which is effective for whole-mount immunofluorescence microscopy.

A method for measurement of the aberration status from high-resolution dark-field images is developed using scanning transmission electron microscopy (STEM), called the Segmented Image Autocorrelation function Matrix (SIAM). The method employs an autocorrelation function from the segmented area in the defocused STEM images from an aligned crystalline specimen to measure the defocus and twofold astigmatism for the probe-forming system. The values measured using this method can be fed directly back to the instrument by changing the strength of the stigmator and the objective lens of the microscope. It is successfully demonstrated that the feedback system can automatically correct the defocus and twofold astigmatism of the microscope after several iterations using practical STEM images from an actual crystalline specimen.

Atomic-size vortex beams have great potential in probing the magnetic moment of materials at atomic scales. However, the limited depth of field of vortex beams constrains the probing depth in which the helical phase front is preserved. On the other hand, electron channeling in crystals can counteract beam divergence and extend the vortex beam without disrupting its topological charge. Specifically, in this article, we report that atomic vortex beams with topological charge ±1 can be coupled to the 2p columnar bound states and propagate for more than 50 nm without being dispersed and losing its helical phase front. We give numerical solutions to the 2p columnar orbitals and tabulate the characteristic size of the 2p states of two typical elements, Co and Dy, for various incident beam energies and various atomic densities. The tabulated numbers allow estimates of the optimal convergence angle for maximal coupling to 2p columnar orbital. We have also developed analytic formulae for beam energy, convergence angle, and hologram-dependent scaling for various characteristic sizes. These length scales are useful for the design of pitch-fork apertures and operations of microscopes in the vortex-beam imaging mode.

Vascular aging is associated with progressive deterioration of vascular homeostasis, a phenomenon that affects organs in different extent. The small vessels of the corpus cavernosum (CC) are very sensitive to functional and structural changes, and particularly susceptible to age-related modifications and atherosclerotic occlusion. Penile erection is a vascular event largely regulated by the relaxation of arterial and corporal smooth muscle (SM) that directly relates to its content and integrity, and inversely to that of connective tissue (CT). Corpora expansion also depends on nitric oxide (NO) bioavailability, a molecule with recognized vasodilatory and antifibrotic properties. Thus, endothelial dysfunction mainly provoked by NO loss is currently considered equivalent to vasculogenic erectile dysfunction (ED), and the earliest clinical manifestation of atherosclerosis and systemic vascular disease. Collagen deposition in tissues, such as CC and heart, strongly compromises their function and is one of the causes of ED and cardiac failure, respectively, but as far as we know no correlation between peripheral and cardiac endothelial dysfunction has been already demonstrated. Therefore, we aimed to evaluate morphological structure and collagen deposition in rat CC, myocardium and tail artery during aging, under high-fat diet (HFD) consumption (a strong contributor to endothelial dysfunction onset), and energy restriction.

Normal cardiac function is maintained through dynamic interactions of cardiac cells with each other and with the extracellular matrix. These interactions are important for remodeling during cardiac growth and pathophysiological conditions. However, the precise mechanisms of these interactions remain unclear. In this study we examined the importance of desmoplakin (DSP) in cardiac cell-cell interactions. Cell-cell communication in the heart requires the formation and preservation of cell contacts by cell adhesion junctions called desmosome-like structures. A major protein component of this complex is DSP, which plays a role in linking the cytoskeletal network to the plasma membrane. Our laboratory previously generated a polyclonal antibody (1611) against the detergent soluble fraction of cardiac fibroblast plasma membrane. In attempting to define which proteins 1611 recognizes, we performed two-dimensional electrophoresis and identified DSP as one of the major proteins recognized by 1611. Immunoprecipitation studies demonstrated that 1611 was able to directly pulldown DSP. We also demonstrate that 1611 and anti-DSP antibodies co-localize in whole heart sections. Finally, using a three-dimensional in vitro cell-cell interaction assay, we demonstrate that 1611 can inhibit cell-cell interactions. These data indicate that DSP is an important protein for cell-cell interactions and affects a variety of cellular functions, including cytokine secretion.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

While negative staining can provide detailed, two-dimensional images of biological structures, the potential of combining tomography with negative staining to provide three-dimensional views has yet to be fully realized. Basic requirements of a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain does not degrade or rearrange with the intensive electron dose (∼106 e/nm2) needed to collect a full set of tomographic images. A commercially available, tungsten-based stain appears to satisfy these prerequisites. Comparison of the surface structure of negatively stained influenza A virus with previous structural results served to evaluate this negative stain. The combination of many projections of the same structure yielded detailed images of single proteins on the viral surface. Corresponding surface renderings are a good fit to images of the viral surface derived from cryomicroscopy as well as to the shapes of crystallized surface proteins. Negative stain tomography with the appropriate stain yields detailed images of individual molecules in their normal setting on the surface of the influenza A virus.

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

The objective of this article was to investigate the effect of carbide and polymer burs caries removal methods on the bond strength of different adhesives to dentin. Resin restorations were performed in sound and caries-affected dentin, after using polymer or carbide burs and bonding with four different adhesive (Single bond, SB; Clearfil SE bond, SEB; FL-Bond II, FLB; and Fuji II-LC, FUJI). Microtensile bond strength (MTBS) was measured. Data were analyzed with ANOVA and Student-Newman-Keuls tests. Debonded surfaces were observed by scanning electron microscopy. Bonded interfaces were examined using light microscopy (Masson's trichrome staining). In sound dentin, MTBS was similar for SEB and SB, and higher than that of FLB and FUJI. Bond strength to carbide bur prepared dentin was similar for SB, SEB, and FLB; FUJI presented the lowest. SB applied on polymer bur excavated dentin presented similar values to those of the carbide bur group; MTBS attained by SEB, FLB, and FUJI decreased when bonding to dentin treated with polymer burs; FUJI yielded pretesting failures in all specimens. Polymer burs created a thick smear layer that was not infiltrated by tested self-etching agents. The bonding effectiveness of self-etching and glass-ionomer-like adhesives to dentin decreased when polymer burs were used.

Color centers in selected micro- and nanodiamond samples were investigated by cathodoluminescence (CL) microscopy and spectroscopy at 298 K [room temperature (RT)] and 77 K [liquid-nitrogen temperature (LNT)] to assess the value of the technique for astrophysics. Nanodiamonds from meteorites were compared with synthetic diamonds made with different processes involving distinct synthesis mechanisms (chemical vapor deposition, static high pressure high temperature, detonation). A CL emission peak centered at around 540 nm at 77 K was observed in almost all of the selected diamond samples and is assigned to the dislocation defect with nitrogen atoms. Additional peaks were identified at 387 and 452 nm, which are related to the vacancy defect. In general, peak intensity at LNT at the samples was increased in comparison to RT. The results indicate a clear temperature—dependence of the spectroscopic properties of diamond. This suggests the method is a useful tool in laboratory astrophysics.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

The charge-coupled devices used in electron microscopy are coated with a scintillating crystal that gives rise to a severe modulation transfer function (MTF). Exact knowledge of the MTF is imperative for a good correspondence between image simulation and experiment. We present a practical method to measure the MTF above the Nyquist frequency from the beam blocker's shadow image. The image processing has been fully automated and the program is made public. The method is successfully tested on three cameras with various beam blocker shapes.

Cord formation in Mycobacterium tuberculosis has been traditionally related to virulence since avirulent M. tuberculosis strains do not form cords. These structures consist of bacilli aligned in parallel, end to end, and side to side, along the long axis of the cord. However, similar structures were recently found in non-pathogenic and in opportunistic Mycobacterium species such as M. abscessus, M. chubuense, M. gilvum, M. haemophilum, M. marinum, M. obuense, M. parafortuitum, and M. vaccae. Although the visualization of cords has been traditionally achieved using Ziehl-Neelsen staining, the resolution of light microscopy does not allow to distinguish among cords, pseudocords, clumps or loose aggregates. Nevertheless, the observation of these formations at high resolution is crucial for two essential applications. Firstly, to identify the unknown mycobacterial components responsible for cording formation, an important step to understand virulence. Secondly, to demonstrate that cording is not specific of M. tuberculosis, since many clinical mycobacteriology laboratories base the tuberculosis diagnostic on the observation of cording morphology, and may mistakenly identify non-tuberculous mycobacteria as M. tuberculosis. Thus, patients would be exposed to an ineffective drug regimen with many adverse side effects. Here, we focused on sample treatment in order to certainly observe cords by using scanning electron microscopy (SEM).

An accurate determination of specimen thickness is essential for quantitative analytical electron microscopy. Here we demonstrate that a position-averaged incoherent bright-field signal recorded on an absolute scale can be used to determine the thickness of on-axis crystals with a precision of ±1.6 nm. This method measures both the crystalline and the noncrystalline parts (surface amorphous layers) of the sample. However, it avoids the systematic error resulting from surface plasmon contributions to the inelastic mean-free-path thickness estimated by electron energy loss spectroscopy.

Mineralized dental tissues and dental pulp were characterized using advanced analytical transmission electron microscopy (TEM) methods. Quantitative X-ray energy dispersive spectroscopy was employed to determine the Ca/P and Mg/P concentration ratios. Significantly lower Ca/P concentration ratios were measured in peritubular dentine compared to intertubular dentine, which is accompanied by higher and variable Mg/P concentration ratios. There is strong evidence that magnesium is partially substituting calcium in the hydroxyapatite structure. Electron energy-loss near-edge structures (ELNES) of C-K and O-K from enamel and dentine are noticeably different. We observe a strong influence of beam damage on mineralized dental tissues and dental pulp, causing changes of the composition and consequently also differences in the ELNES. In this article, the importance of TEM sample preparation and specimen damage through electron irradiation is demonstrated.

Understanding cytoskeletal dynamics in living tissue is prerequisite to understanding mechanisms of injury, mechanotransduction, and mechanical signaling. Real-time visualization is now possible using transfection with plasmids that encode fluorescent cytoskeletal proteins. Using this approach with the muscle-specific intermediate filament protein desmin, we found that a green fluorescent protein–desmin chimeric protein was unevenly distributed throughout the muscle fiber, resulting in some image areas that were saturated as well as others that lacked any signal. Our goal was to analyze the muscle fiber cytoskeletal network quantitatively in an unbiased fashion. To objectively select areas of the muscle fiber that are suitable for analysis, we devised a method that provides objective classification of regions of images of striated cytoskeletal structures into “usable” and “unusable” categories. This method consists of a combination of spatial analysis of the image using Fourier methods along with a boosted neural network that “decides” on the quality of the image based on previous training. We trained the neural network using the expert opinion of three scientists familiar with these types of images. We found that this method was over 300 times faster than manual classification and that it permitted objective and accurate classification of image regions.

Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an “in vivo cryotechnique” (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.

The 49 type I natural pink diamonds examined exhibit color restricted to lamellae or bands oriented along {111} that are created by plastic deformation. Pink diamonds fall into two groups: (1) diamonds from Argyle in Australia and Santa Elena in Venezuela are heavily strained throughout and exhibit pink bands alternating with colorless areas, and (2) diamonds from other localities have strain localized near the discrete pink lamellae. Growth zones are highlighted by a blue cathodoluminescence (CL) and crosscut by the pink lamellae that emit yellowish-green CL that originates from the H3 center. This center probably forms by the recombination of nitrogen-related centers (A-aggregates) and vacancies mobilized by natural annealing in the Earth's mantle. Twinning is the most likely mechanism through which plastic deformation is accommodated for the two groups of diamonds. The plastic deformation creates new centers visible through spectroscopic methods, including the one responsible for the pink color, which remains unidentified. The differences in the plastic deformation features, and resulting CL properties, for the two groups might correlate to the particular geologic conditions under which the diamonds formed; those from Argyle and Santa Elena are deposits located within Proterozoic cratons, whereas most diamonds originate from Archean cratons.