Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Using confocal Raman microscopy to real‐time monitor poplar cell wall swelling and dissolution during ionic liquid pretreatment

The ionic liquids (ILs) are recognized as the potential solvents for the pretreatment of lignocellulosic materials before biomass conversion. However, little knowledge of how the cell wall of biomass responds to the IL locally and dynamically during the pretreatment is available. In the current work, the process of IL pretreatment of poplar using 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) was real‐time monitored on a cellular level by employing confocal Raman microscopy. The results showed that the biomass dissolution during the IL pretreatment can be clearly divided into two stages: (1) slow penetration of IL, and (2) rapid dissolution of lignin and carbohydrates. In this case, the onset of the dissolution of these compositions occurred only after the cell wall of biomass swelled to a certain extent. Because the first stage was a slow process which determined the process reaction rate, it can be deduced that enhancing the penetration capacity of IL was crucial for improving the pretreatment efficiency. Based on the obtained results, a model was proposed to better understand how the plant cell wall responds to the IL before, during, and after pretreatment. Microsc. Res. Tech. 77:609–618, 2014. © 2014 Wiley Periodicals, Inc.     

Traditional coating techniques in scanning electron microscopy compared to uncoated charge compensator technology: Looking at human blood fibrin networks with the ZEISS ULTRA Plus FEG‐SEM

Scanning electron microscopy (SEM) studies surface morphology. Biological material needs to be coated to render the material conductive, and gold coating is traditionally used, although other coating material like carbon and ruthenium vapors may also be used. With modern SEM technology (e.g., ZEISS ULTRA Plus FEG‐SEM), we are able to work at very low kilovolts and also view fine surface structure in much better detail than with previous older technology. Some machines also allow for the study of uncoated material, although this is usually not done with biological material. This study focuses on surface clarity by comparing gold, ruthenium vapor, and carbon coating techniques for biological material. Human fibrin networks are used as example. Uncoated specimens are also viewed with a ZEISS ULTRA Plus FEG‐SEM because of its unique nitrogen charge compensator, and here, the first micrographs for uncoated human fibrin networks versus carbon, gold, and ruthenium coating are shown. We conclude that gold coating for biological material is not preferable with the latest SEM machines, as this method forms gold islands on top of the biological material and therefore produces a false surface morphology. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

F‐actin and α‐actinin reorganization mediates initial fibroblast interaction with CoCr alloy particles in vitro

Asceptic loosening remains the primary cause for failure of joint implant. The active role of fibroblasts in mediating asceptic loosening is however not well documented. In this study the initial interactions of fibroblasts with metal particles was studied by evaluating changes in the cytoskeletal structure and cytokine level. Murine L929 fibroblasts cultured with cobalt chromium particles were observed by phase contrast and scanning electron microscopy (SEM). Changes in the cytoskeletal rearrangement of F‐actin and α‐actinin focal adhesion plaques were studied by confocal microscopy. Expression of the proinflammatory cytokines IL‐6 and IL‐1α were analyzed by ELISA. The role of actin filaments and microtubules in particle uptake were determined at low temperature and in presence of colchicine and cytochalasin B. Phase contrast and SEM studies reveal that the metal particles adhere to the fibroblasts. The cellular cytoplasm was observed to grow over the particles and is suggestive of particle uptake. Confocal microscopy shows the presence of voids within the F‐actin cytoskeletal framework corresponding to areas occupied by the metal particles, indicating the possible uptake of these particles. Aggregates of α‐actinin into patches at the cell surface were also noted. Adherence and uptake of particles did not occur at low temperature and in presence of cytochalasin B, indicating that it is an active energy‐dependent process involving actin filaments. Changes in the levels of cytokine IL‐6 and IL‐1α were not observed suggesting the role of other cytokine molecules in mediating the inflammatory response to wear debri by fibroblasts. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

MRT Letter: High resolution SEM imaging of nano‐architecture of cured urea‐formaldehyde resin using plasma coating of osmium

Nanoarchitecture of cured urea‐formaldehyde (UF) resins was examined with a field‐emission scanning electron microscope (FE‐SEM) after coating samples with osmium, which is considered to produce particles of considerably smaller size compared to other metal coatings used in SEM studies. This method enabled comparison of the nanoarchitecture of UF resins of low (1.0) and high (1.6) formaldehyde/urea (F/U) mole ratios to be made, based on imaging of extremely small size particles as part of UF resin architecture, not described before. Imaging revealed presence of relatively large globular particles (148.084–703.983 nm size range) as well as smaller substructures (28.004–39.604 nm size range) as part of the architecture of 1.0‐mole UF resin. Globular particles were also present in 1.6 mole UF resin, but of considerably smaller size (14.760–50.269 nm). The work presented demonstrates usefulness of osmium coating in unraveling the intricacies of the nanostructural organization of cured UF resins, prompting wider application of this immensely useful but grossly underutilized metal coating type in high resolution SEM examination of biological and materials samples. Microsc. Res. Tech. 76:1108–1111, 2013. © 2013 Wiley Periodicals, Inc.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Using transmission Kikuchi diffraction to characterise α variants in an α+β titanium alloy

Two phase titanium alloys are important for high‐performance engineering components, such as aeroengine discs. The microstructures of these alloys are tailored during thermomechanical processing to precisely control phase fractions, morphology and crystallographic orientations. In bimodal two phase (α + β) Ti‐6Al‐2Sn‐4Zr‐2Mo (Ti‐6242) alloys there are often three microstructural lengthscales to consider: large (～10 μm) equiaxed primary α; >200 nm thick plate α with a basketweave morphology; and very fine scaled (<50 nm plate thickness) secondary α that grows between the larger α plates surrounded by retained β. In this work, we utilise high spatial resolution transmission Kikuchi diffraction (TKD, also known as transmission‐based electron backscatter diffraction, t‐EBSD) and scanning electron microscopy (SEM)‐based forward scattering electron imaging to resolve the structures and orientations of basketweave and secondary α in Ti‐6242. We analyse the α variants formed within one prior β grain, and test whether existing theories of habit planes of the phase transformation are upheld. Our analysis is important in understanding both the thermomechanical processing strategy of new bimodal two‐phase titanium alloys, as well as the ultimate performance of these alloys in complex loading regimes such as dwell fatigue. Our paper champions the significant increase in spatial resolution afforded using transmission techniques, combined with the ease of SEM‐based analysis using conventional electron backscatter diffraction (EBSD) systems and forescatter detector (FSD) imaging, to study the nanostructure of real‐world engineering alloys.     

High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

A longstanding limitation of imaging with serial block‐face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block‐face due to image jitter. Typically, variable‐pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal‐to‐noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block‐face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block‐face ultramicrotome. This system enables the application of nitrogen gas precisely over the block‐face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high‐resolution block‐face imaging of even the most charge prone of epoxy‐embedded biological samples.     

New application of variable‐pressure/environmental microscopy to semiconductor inspection and metrology

Variable‐pressure/environmental scanning electron microscopy has been used for successful investigation binary and phase‐shifting chromium on quartz optical photomasks. This methodology was also applied to patterned 193 nm photoresist structures. The application of this methodology to semiconductor metrology is new because of the recent availability of variable‐pressure scanning electron microscopy (SEM) instrumentation equipped with high‐resolution, high‐signal, thermally assisted field emission technology in conjunction with large chamber and sample transfer capabilities. The variable‐pressure SEM methodology employs a gaseous environment around the sample to help diminish the charge build‐up that occurs under irradiation with the electron beam. Although very desirable for the charge reduction in many biological, pharmaceutical, and food applications, this methodology has not been employed for semiconductor photomask or wafer metrology until now. This is a new application of this technology to this area, and it shows great promise in inspection, imaging, and metrology in a charge‐free operational mode. For accurate metrology, variable‐pressure SEM methodology also affords a path that minimizes, if not eliminates, the need for charge modeling. This paper presents some of the early results in the variable‐pressure SEM metrology of photomask and photoresist structures.     

Surface roughness and filler particles characterization of resin‐based composites

The purpose of this study was to evaluate the surface roughness (Ra), and the morphology and composition of filler particles of different composites submitted to toothbrushing and water storage. Disc‐shaped specimens (15 mm × 2 mm) were made from five composites: two conventional (Z100?, and Filtek? Supreme Ultra Universal, 3M), one “quick‐cure” (Estelite ∑ Quick, Tokuyama), one fluoride‐releasing (Beautiful II, Shofu), and one self‐adhering (Vertise Flow, Kerr) composite. Samples were finished/polished using aluminum oxide discs (Sof‐Lex, 3M), and their surfaces were analyzed by profilometry (n = 5) and scanning electron microscopy (SEM; n = 3) at 1 week and after 30,000 toothbrushing cycles and 6‐month water storage. Ra data were analyzed by two‐way analysis of variance and Tukey's test (α = 0.05). Filler particles morphology and composition were analyzed by SEM and X‐ray dispersive energy spectroscopy, respectively. Finishing/polishing resulted in similar Ra for all the composites, while toothbrushing and water storage increased the Ra of all the tested materials, also changing their surface morphology. Beautifil II and Vertise Flow presented the highest Ra after toothbrushing and water storage. Filler particles were mainly composed of silicon, zirconium, aluminum, barium, and ytterbium. Size and morphology of fillers, and composition of the tested composites influenced their Ra when samples were submitted to toothbrushing and water storage.     

Backscattered electron image of osmium‐impregnated/macerated tissues as a novel technique for identifying the cis‐face of the Golgi apparatus by high‐resolution scanning electron microscopy

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three‐dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis‐ or trans‐faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis‐Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis‐elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron‐mode SEM. Prolonged osmication by osmium impregnation (2% OsO₄ solution at 40°C for 40 h) and osmium maceration (0.1% OsO₄ solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.     

Imaging connected porosity of crystalline rock by contrast agent‐aided X‐ray microtomography and scanning electron microscopy

We set out to study connected porosity of crystalline rock using X‐ray microtomography and scanning electron microscopy with energy dispersive X‐ray spectroscopy (SEM‐EDS) with caesium chloride as a contrast agent. Caesium is an important radionuclide regarding the final deposition of nuclear waste and also forms dense phases that can be readily distinguished by X‐ray microtomography and SEM‐EDS. Six samples from two sites, Olkiluoto (Finland) and Grimsel (Switzerland), where transport properties of crystalline rock are being studied in situ, were investigated using X‐ray microtomography and SEM‐EDS. The samples were imaged with X‐ray microtomography, immersed in a saturated caesium chloride (CsCl) solution for 141, 249 and 365 days and imaged again with X‐ray microtomography. CsCl inside the samples was successfully detected with X‐ray microtomography and it had completely penetrated all six samples. SEM‐EDS elemental mapping was used to study the location of caesium in the samples in detail with quantitative mineral information. Precipitated CsCl was found in the connected pore space in Olkiluoto veined gneiss and in lesser amounts in Grimsel granodiorite. Only a very small amount of precipitated CsCl was observed in the Grimsel granodiorite samples. In Olkiluoto veined gneiss caesium was found in pinitised areas of cordierite grains. In the pinitised areas caesium was found in notable excess compared to chloride, possibly due to the combination of small pore size and negatively charged surfaces. In addition, elevated concentrations of caesium were found in kaolinite and sphalerite phases. The findings concerning the location of CsCl were congruent with X‐ray microtomography.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

SEM and AFM studies of dip‐coated CuO nanofilms

Cupric oxide (CuO) semiconducting thin films were prepared at various copper sulfate concentrations by dip coating. The copper sulfate concentration was varied to yield films of thicknesses in the range of 445–685 nm by surface profilometer. X‐ray diffraction patterns revealed that the deposited films were polycrystalline in nature with monoclinic structure of (?111) plane. The surface morphology and topography of monoclinic‐phase CuO thin films were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. Surface roughness profile was plotted using WSxM software and the estimated surface roughness was about ～19.4 nm at 30 mM molar concentration. The nanosheets shaped grains were observed by SEM and AFM studies. The stoichiometric compound formation was observed at 30 mM copper sulfate concentration prepared film by EDX. The indirect band gap energy of CuO films was increased from 1.08 to 1.20 eV with the increase of copper sulfate concentrations. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Influence of acceleration setting reaction by halogen light‐curing on GIC–dentin interface: Qualitative analysis by SEM

The aim of this study was to evaluate by scanning electron microscope (SEM) photomicrographs the influence of application of halogen light‐curing for fastening the set reaction of high‐viscosity glass ionomer cements (GIC) by assessing the material/dentin interface. Twelve human primary canines were assigned in four groups (n = 3) according to the GIC (Fuji IX, GC or Maxxion R, FGM) and application of halogen light‐curing (60 sec or control—no external energy). Blocks with approximately 6 mm of height were buildup on previously pre‐treated dentin surface in according to the experimental group. After storage at 37 °C, 100% humidity for 48 h, the specimens were then sectioned in slices with 1‐mm thick. The slices were qualitative analyzed using SEM to evaluate possible structural changes. Two examiners independently evaluated the images in order to observe the spherical hollow spaces of each tooth. The photomicrographs revealed the presence of spherical hollow spaces in all experimental groups. However, in both groups that received halogen light‐curing application, it was possible to observe that the presence of these hollow spaces decreased in size and quantity. It can be concluded that the halogen light‐curing application positively decreases in size and quantity in the presence of spherical hollow spaces in GIC.     

Surface micromorphology of dental composites [CE‐TZP]‐[Al2O3] with Ca+2 modifier

The objective of this study was to characterize the three‐dimensional (3D) surface micromorphology of the ceramics produced from nanoparticles of alumina and tetragonal zirconia (t‐ZrO₂) with addition of Ca⁺² for sintering improvement. The 3D surface roughness of samples was studied by atomic force microscopy (AFM), fractal analysis of the 3D AFM‐images, and statistical analysis of surface roughness parameters. Cube counting method, based on the linear interpolation type, applied for AFM data was used for fractal analysis. The morphology of non‐modified ceramic sample was characterized by the rather big (1–2 μm) grains of α‐Al₂O₃ phase with a habit close to hexagonal drowned in solid solution of t‐ZrO₂ with smooth surface. The pattern surfaces of modified composite content a little amount of elongated prismatic grains with composition close to the phase of СаСеAl₃О₇ as well as hexahedral α‐Al₂O₃‐grains. Fractal dimension, D, as well as height values distribution have been determined for the surfaces of the samples with and without modifying. It can be concluded that the smoothest surface is of the modified samples with Ca⁺² modifier but the most regular one is of the non‐modified samples. A connection was observed between the surface morphology and the physical properties as assessed in previous works. Microsc. Res. Tech. 78:840–846, 2015. © 2015 Wiley Periodicals, Inc.     

Two‐photon laser scanning microscopy as a useful tool for imaging and evaluating macrophage‐, IL‐4 activated macrophage‐ and osteoclast‐based In Vitro degradation of beta‐tricalcium phosphate bone substitute material

Two‐photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two‐photon laser scanning microscopy. Beta‐tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin‐4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta‐tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL‐4 activated macrophages. An average of ～32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL‐4 activated macrophages. For the first time by applying two‐photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143–152, 2014. © 2013 Wiley Periodicals, Inc.     

Morphological investigation of various orthodontic lingual bracket slots using scanning electron microscopy and atomic force microscopy

Various labial and lingual orthodontic appliances with aesthetic materials have been developed due to an increased demand in aesthetic orthodontic treatment. However, there are few reports regarding the morphology of lingual orthodontic appliances. Therefore, this study evaluates the roughness of slot surfaces of various orthodontic lingual brackets using field emission scanning electron microscopy (FE‐SEM) and atomic force microscopy (AFM). Three types of stainless steel lingual brackets (Stealth^®, 7th Generation^®, and Clippy L^®) and one gold lingual bracket (Incognito?) with a slot size of 0.018 inches × 0.025 inches (0.457 × 0.635 mm²) were selected as representative lingual materials. Both FE‐SEM and AFM examinations showed that the Stealth^® and Clippy L^® brackets had the lowest surface roughness, while the 7th Generation^® bracket had the highest surface roughness. There was a significant difference in surface morphology between the types of lingual brackets, even when composed of the same material. The surface roughness of the bracket slot was dependent on the manufacturing process or surface polishing process rather than the fundamental properties of the bracket materials. There was no significant difference in the mean surface roughness of the slot floor between gold and stainless steel lingual brackets. These findings suggest that, although the gold lingual bracket is very expensive, it has great potential for use in patients with nickel allergy.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

Atmospheric scanning electron microscopy and its applications for biological specimens

Scanning electron microscopy in ambient conditions (Air‐SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air‐SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)‐SEM. Sharpness was lower than that of FE‐SEM, but foot process of podocytes was observed at high magnification. Air‐SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air‐SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air‐SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.