Palyno‐anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy

Palyno‐anatomical study of monocots taxa using Light and Scanning Electron Microscopy (SEM) was first time conducted with a view to evaluating their taxonomic significance. Studied plants were collected from different eco‐climatic zones of Pakistan ranges from tropical, sub‐tropical, and moist habitats. The aim of this study is to use palyno‐anatomical features for the correct identification, systematic comparison, and investigation to elucidate the taxonomic significance of these features, which are useful to taxonomists for identifying monocot taxa. A signification variation was observed in quantitative and qualitative characters by using the standard protocol of light microscopy (LM) and SEM. Epidermal cell length varied from maximum in Allium griffthianum (480 ± 35.9) μm at the adaxial surface to minimum in Canna indica (33.6 ± 8.53) μm on abaxial surface. Maximum exine thickness was observed in Canna indica (4.46) μm and minimum in Allium grifthianum (0.8) μm. Variation was observed in shape and exine ornamentation of the pollen, shape of the epidermal cell, number, size, and type of stomata, guard cell shape, and anticlinal wall pattern. Based on these palyno‐anatomical features a taxonomic key was developed, which help in the discrimination of studied taxa. In conclusion, LM and SEM pollen and epidermal morphology is explanatory, significant, and can be of special interest for the plant taxonomist in the correct identification of monocots taxa.     

Histometric and histomorphologic comparison of combustion and ambustion using in vivo reflectance‐confocal microscopy

Background: When combustion and ambustion induce a superficial injury, they are summarized as superficial burns, regardless of the underlying cause. Reflectance‐confocal microscopy (RCM) allows noninvasive imaging of the human skin on morphological features. We hypothesized that combustion and ambustion have different histomorphological effects on the human skin. Methods: Superficial burns caused by combustion (CO‐group, five females, three males; aged 26.8 ± 14.2 years) and caused by ambustion (AM‐group, four females, four males; aged 28.1 ± 13.8 years) were evaluated 24 h after injury. The following parameters were obtained using RCM on injured and noninjured (control) site: horny layer thickness, epidermal thickness, granular cell size, basal layer thickness. Results: Compared with the controls (12.8 ± 2.5 μm), horny layer thickness decreased significantly to 10.6 ± 2.1 μm in the CO‐group, whereas it increased significantly to 17.8 ± 2.8 μm in the AM‐group. The epidermal thickness did not differ significantly in CO‐group (47.9 ± 2.1 μm) and AM‐group (49.0 ± 3.1 μm), however, both increased significantly compared with the controls (42.7 ± 1.6 μm). The basal layer thickness increased more in AM‐group (17.0 ± 1.2 μm) compared to CO‐group (15.4 ± 1.1 μm). Both differed significantly compared with their controls (13.9 ± 0.9 μm). The granular cell size increased significantly in both groups ompared to the controls (721 ± 42 μm), however, a significantly higher increase was observed in CO‐group compared to AM‐group (871 ± 55 μm vs. 831 ± 51 μm). Conclusions: RCM evaluates significant histomorphological differences in superficial burns caused by combustion and ambustion. The term “superficial burn” should consider the underlying cause and thus supplemented by the term “combustion” or “ambustion.” Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of Albanian water frog,Pelophylax shqipericus,sperm traits and morphology,by using phase contrast microscopy

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth‐edged, and solid‐staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 μm ± 0.32, while the tail length resulted 29.24 ± 1.75 μm. The average thickness of the head was shown to be 3.45 μm. The total sperm length resulted to be 41.02 ± 1.83 μm. The average sperm concentration was estimated of 25.5 × 10⁶/ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of “Lessonae” frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.     

Palyno‐morphological investigations of subtropical endangered flora of Capparidaceae through light and scanning electron microscopy

The research was performed to investigate pollen morphology of endangered species of Capparidaceae in subtropical regions of Pakistan. The distinguishing characters were investigated by using light microscope and scanning electron microscope. Palynological study is comprised of pollen shape, pollen type, exine sculpturing, polar and equatorial diameter, length and width of colpi, mesocolpium, and exine thickness. In polar view, Cleome viscosa exhibited the highest pollen size 26.4 (32.7–24.5 μm) ±0.776 whereas Capparis spinosa appeared to be the lowest 12.6 (14.5?10.7 μm) ±0.400. In equatorial view, Cleome viscosa had the largest pollen size 17.1 (20.0–15.0 μm) ±0.606 and Capparis spinosa had the smallest pollen size 9.7 (12.50–8.00 μm) ±0.394. The maximum fertility percentage has been observed in Capparis spinosa, that is, 98.96% and minimum in Cleome viscosa, that is, 82.93%. Diagnostic key has been constructed to state the essential diagnostic features by means of which the taxa can be identified. Remarkable variations have been observed in pollen size, shape, and exine sculpturing. All the selected species were tricolporate. Prolate to subprolate pollen were observed. There is a great variation existed in exine sculpturing such as in Capparis decidua and C. sp. nova sculpturing is reticulate, in Capparis himalayensis sculpturing is Scabrate granulate, in Capparis spinosa sculpturing is Psilate, in Cleome viscosa sculpturing is regulate‐reticulate, in Dipterygium glaucum sculpturing is regulate and in Gynandropsis gynandra sculpturing is striate‐regulate. On the basis of overall characteristics of pollen it seems that palynology of this family is helpful at the generic and specific level.     

Reflectance confocal‐laser‐scanning microscopy in vivo assessments of cigarette‐induced dynamic alterations of cutaneous microcirculation on histomorphological level

Objective: Until now, high resolution reflectance confocal‐laser‐scanning microscopy (CLSM) was used for observation of cutaneous morphology in vivo and in real time. We hypothesized that CLSM also allows observation of dynamic processes of cutaneous microcirculation. Methods: Reflectance CLSM (Vivascope1500; Lucid, Rochester, NY) was performed in 24 young male habitual smokers (23 years, range: 19–26, body mass index 23.9 ± 4.04) with relatively limited cigarette exposure (mean: 3.1 ± 2.4 pack‐years). Eight matched nonsmokers served as controls. The quantitative blood cell flow and the diameter of capillary loops were determined prior (baseline), during, as well as 5 and 10 min after smoking. Results: Baseline value for blood cell flow was 55.50 ± 2.33 cells/min, and decreased over 45% during smoking (30.43 ± 3.76/min; P = 0.02). They were still 22% lower (43.33 ± 2.45/min; P = 0.01) 5 min after smoking and exceeded baseline values 10 min after smoking by 13% (63.00 ± 3.10/min; P > 0.05). The baseline values for capillary loop diameter (9.03 ± 0.22 μm) decreased by 21% (7.18 ± 0.28 μm; P = 0.03) during smoking, remained about 9% (8.23 ± 0.18 μm; P = 0.01) lower 5 min after smoking and exceeded baseline values insignificantly by 4% (9.38 ± 0.28 μm; P > 0.05) 10 min after smoking. There were no significant differences to the controls. Conclusion: Reflectance CLSM enables qualitative and quantitative observation of dynamic processes of cutaneous microcirculation on histomorphological level. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Characterization of dimensions of ellipsoidal microparticles via electron microscopy

The analysis and the use of metallic shadow casting and specimen tilting for accurately determining the dimensions of microellipsoids by electron microscopy (EM) are presented. The procedure uses the direct image and two different shadows or two different specimen tilts to unambiguously determine the dimensions of an ellipsoid. A novel computer-aided matching procedure is used to determine dimensions and orientations. The analysis was applied to a sample of polymer prolate microspheroids, which have recently been synthesized in our laboratory. The shape of the particles was confirmed and their dimensions and orientations were determined. The particles were shown to have a length (twice the principal axis) of 3.30±0.02 μm and a diameter (twice the minor axis) of 1.66±0.04 μm. Thus, both EM techniques were demonstrated to be practical and precise for determining dimensions of individual microellipsoids.     

Nitric oxide regulates mitochondrial re‐modelling in interscapular brown adipose tissue: ultrastructural and morphometric‐stereologic studies

As a complex, cell‐specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of l ‐arginine‐nitric oxide (NO)‐producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving l ‐arginine, a substrate of NO synthases (NOSs), or N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub‐groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. l ‐Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold‐induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in l ‐arginine‐treated rats. Ultrastructural examinations of l ‐arginine‐treated cold‐acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well‐ordered cristae in their profiles), which however were inactive in l ‐arginine‐receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, l ‐NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.     

Time‐lapse microscopy using smartphone with augmented reality markers

A prototype system that replaces the conventional time‐lapse imaging in microscopic inspection for use with smartphones is presented. Existing time‐lapse imaging requires a video data feed between a microscope and a computer that varies depending on the type of image grabber. Even with proper hardware setups, a series of tedious and repetitive tasks is still required to relocate to the region‐of‐interest (ROI) of the specimens. In order to simplify the system and improve the efficiency of time‐lapse imaging tasks, a smartphone‐based platform utilizing microscopic augmented reality (μ‐AR) markers is proposed. To evaluate the feasibility and efficiency of the proposed system, a user test was designed and performed, measuring the elapse time for a trial of the task starting from the execution of the application software to the completion of restoring and imaging of an ROI saved in advance. The results of the user test showed that the average elapse time was 65.3 ± 15.2 s with 6.86 ± 3.61 μm of position error and 0.08 ± 0.40 degrees of angle error. This indicates that the time‐lapse imaging task was accomplished rapidly with a high level of accuracy. Thus, simplification of both the system and the task was achieved via our proposed system. Microsc. Res. Tech. 77:243–249, 2014. © 2014 Wiley Periodicals, Inc.     

Capturing the elasticity and morphology of live fibroblast cell cultures during degradation with atomic force microscopy

Atomic force microscopy, in a liquid environment, was used to capture in vitro the morphological and mechanical changes that cultured fibroblasts undergo as time elapses from the completion of the cell culture. Topography images illustrated that initially, the nucleus had a height of 1.18 ± 0.2 μm, and after 48 h it had decreased to 550 ± 60 nm; similarly, the cell membrane exhibited significant shrinkage from 34 ± 4 to 23 ± 2 μm. After each image scan, atomic force microscopy indentation was performed on the centre of the nucleus, to measure the changes in the cell elasticity. Examination of the force‐distance curves indicated that the membrane elastic modulus at the nucleus remained the same within the time frame of 48 h, even though the cell morphology had significantly changed.     

The changes of gap junctions between pituitary folliculo‐stellate cells during the postnatal development of zucker fatty and lean rats

We investigated the effect of leptin on the postnatal development of gap junctions between folliculo‐stellate cells by using Zucker fatty (fa/fa) rats that have defects of the functional leptin receptor. Male Zucker fatty rats (fa/fa) and male Zucker lean rats (+/+) were used at each of the following postnatal ages: 20, 30, 40, 50, 60, 70, 80, 90 days, and 1 year. On one of the aforementioned dates, the anterior pituitary glands were prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions, and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicular profile. In Zucker lean male rats, the number of gap junctions remained relatively constant from days 50 to 90 (0.44 ± 0.02 to 0.49 ± 0.03), and was similar in 1 year old rats (0.47 ± 0.03). These data were statistically higher compared to Zucker fatty male rats. In Zucker fatty male rats, very few gap junctions were observed in 30‐day‐old rats (0.04 ± 0.01: mean ± SE). This disruption of gap junction formation persisted, and the number of gap junctions remained constant and showed a low level from days 40 to 90 (0.11 ± 0.02 to 0.17 ± 0.02); this finding was similar in 1‐year‐old rats (0.17 ± 0.02). These observations indicate that the effect of leptin over the gap junction formation within the anterior pituitary glands was directly mediated by interaction with the functional leptin receptor present on the folliculo‐stellate cells. Microsc. Res. Tech. 77:31–36, 2014. © 2013 Wiley Periodicals, Inc.     

Structural and functional alterations of cellular components as revealed by electron microscopy

Scanning (SEM) and transmission electron microscopy (TEM) are two fundamental microscopic techniques widely applied in biological research for the study of ultrastructural cell components. With these methods, especially TEM, it is possible to detect and quantify the morphological and ultrastructural parameters of intracellular organelles (mitochondria, Golgi apparatus, lysosomes, peroxisomes, endosomes, endoplasmic reticulum, cytoskeleton, nucleus, etc.) in normal and pathological conditions. The study of intracellular vesicle compartmentalization is raising even more interest in the light of the importance of intracellular localization of mediators of the signaling in eliciting different biological responses. The study of the morphology of some intracellular organelles can supply information on the bio‐energetic status of the cells. TEM has also a pivotal role in the determination of different types of programmed cell death. In fact, the visualization of autophagosomes and autophagolysosomes is essential to determine the occurrence of autophagy (and also to discriminate micro‐autophagy from macro‐autophagy), while the presence of fragmented nuclei and surface blebbing is characteristic of apoptosis. SEM is particularly useful for the study of the morphological features of the cells and, therefore, can shed light, for instance, on cell–cell interactions. After a brief introduction on the basic principles of the main electron microscopy methods, the article describes some cell components with the aim to demonstrate the huge role of the ultrastructural analysis played in the knowledge of the relationship between function and structure of the biological objects. Microsc. Res. Tech., 76:1057–1069, 2013. © 2013 Wiley Periodicals, Inc.     

Comparative foliar anatomical and pollen morphological studies of Acanthaceae using light microscope and scanning electron microscope for effective microteaching in community

In this study, foliar anatomy and pollen morphology of 10 species of Acanthaceae has been investigated using light and scanning electron microscopy. The study was aimed to highlight the role of microscopy in microteaching at community for proper characterization of plants using palyno‐anatomical characters including pollen type, exine sculpturing, shape of epidermal cells, pattern of anticlinal wall, type and size of stomata, and trichome. Most of the species have polygonal cell shapes but some species have irregular, tetragonal, and pentagonal shape of epidermal cells. The largest epidermal cell length on adaxial and abaxial surface were observed in Asystasia gangetica 66.95 and 87.40 μm whereas least was observed on adaxial surface in Justicia adhatoda 36.9 μm and on abaxial surface in Barleria cristata 35.65 μm. In anatomy, species have diacytic type of stomata, whereas stomata of paracytic type observed in two species, while in A. gangetica cyclocytic type of stomata are present. Quantitively on abaxial surface, largest stomata length 29.9 μm and width 24.30 μm was noted in B. cristata. While shortest stomata length was observed in Ruellia prostrata 25.95 μm whereas minimum width of stomata was examined in Barleria acanthoides 2.05 μm. The diversity of trichomes are present in all species except in Ruellia brittoniana. Acanthaceae can be characterized by exhibiting different pollen morphology having five types of pollen shapes, prolate, spheroidal, perprolate, subprolate, and oblate spheroidal. Exine peculiarities showing variations such as reticulate, granulate, coarsely reticulate, lophoreticulate, perforate tectate, and granulate surface were examined.     

Functionalized magnetic nanoparticles attenuate cancer cells proliferation: Transmission electron microscopy analysis

The penetration and transportation of nanoparticles (NPs) inside the cancer cells is critical to study. In this article, cancer cells (HCT‐116) were treated with functionalized magnetic NPs for the period of 48 hr and studied their ultrastructure by transmission electron microscopy (TEM). The NPs‐treated cells were prepared by chemical fixation and sliced into electron‐transparent arbitrary sections (200 × 200 μm²) by ultramicrotome. Major events of NPs–cell interaction, such as penetration of NPs, encapsulation of NPs into the intracellular compartments, transportation of NPs, and NPs exit, were examined by TEM to understand the mechanism of cell death. The NPs showed the uniform spherical shape with broad size distribution (100–400 nm), while cells displayed irregular morphology with average diameter ~5 μm. Our results showed the successful penetration of NPs deep into the cell, encapsulation, transportation, and exocytosis. Furthermore, we tested the different concentrations (0, 1.5, 12.5, and 50 μg/ml) of NPs on cancer cells and evaluated the cell viability. Laser confocal microscopy and colorimetric analysis together demonstrated that the cell viability is a dose‐dependent phenomenon, where 50 μg/ml specimen showed the highest killing of cancer cells compared to other dosages.     

Extremely thin layer plastification for focused‐ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells

Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 m² cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Light microscopy of Pakistani Berberis leaf cuticles and its taxonomic implications

Taxonomy of the genus Berberis is quite complex, due to overlapping morphological characters, making it very difficult to differentiate the species within the genus. In order to resolve this taxonomic complexity, the foliar anatomy of 10 Berberis L. species was carried out, for the first time from Pakistan, using light microscopy (LM). Significant variation in terms of epidermal cells shape, size, cell wall pattern, and stomata type was observed. B. baluchistanica has the largest epidermal cells, Adaxial: length = 45–(53.9 ± 3.6)–62.5 μm; and width = 22.5–(26.3 ± 1.3)–30 μm; Abaxial: length = 37.5–(43.25 ± 2.5)–50 μm; and width = 20–(22.6 ± 0.8)–25. The highest number of stomata was observed in B. glaucocarpa as 62 on the abaxial surface while the lowest number of stomata was recorded in B. baluchistanica as 8 on the adaxial surface. Of 10 investigated species, 6 possess anomocytic type stomata, while 2 species that is, B. aitchisonii and B. parkeriana have both anomocytic and anisocytic stomata while B. baluchistanica and B. calliobotrys have only paracytic type stomata. The highest number of cells per unit area was present on the adaxial surface of B. calliobotrys ranging from 245–(252.4)–260 followed by B. parkeriana with 209–(227.8)–250 on the abaxial surface. Stomatal index (SI) also varied considerably and was the lowest (2.6) percentage in B. baluchistanica and highest (31.9) percentage in B. kunawurensis. A taxonomic key based on micro‐morphological characters is provided for species identification.     

Cryo‐scanning electron microscopy and light microscopy for the study of fungi interactions

The application of the cryo‐scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Micro‐CT scouting for transmission electron microscopy of human tissue specimens

Transmission electron microscopy (TEM) provides sub‐nanometre‐scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro‐CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench‐top micro‐CT scanner with 10 μm resolution was used to determine the location of patches of the mucous membrane in osmium‐stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra‐thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.     

Simple technique for high‐throughput marking of distinguishable micro‐areas for microscopy

Today's (nano)‐functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 μm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin‐film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron‐beam evaporation which ensures good line‐of‐sight deposition. This inexpensive high‐throughput method has several advantageous over other marking techniques such as focused ion‐beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion‐beam sectioning to obtain a thin lamellar of a particular pre‐selected area.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.