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.     

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY^® FL C₁₂ (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH₄Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.     

Two‐frequency CARS imaging by switching fiber laser excitation

To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber‐laser source. Stokes pulses were generated by soliton self‐frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid‐crystal retarder. Proof‐of‐principle is demonstrated by coherent anti‐Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm^?1, where polystyrene has a Raman transition, and 2,950 cm^?1, where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes.     

Differentiation of live‐viable versus dead bacterial endospores by calibrated hyperspectral reflectance microscopy

This paper describes the use of hyperspectral imaging microscopy (HIM) for the characterization and differentiation of live viable versus dead/non‐viable bacterial endospores for two species of Bacillus. To accomplish this, endospore‐forming Bacillus were cultured and differentiated into endospores. Non‐viable endospores were produced using sporicidal methods representing standard decontamination procedures incorporating chlorine and peroxide. Finally, endospore samples were lyophilized to prepare them for spectral analysis. Prior to HIM, baseline spectral reflectance characterizing the endospores was measured using an ASD (400–900 nm) reflectance spectrometer. These data were used to calibrate the resulting spectral image data. HIM data comprising 32 images ranging from 400 to 720 nm (visible to near infrared) were recorded using a C‐mounted VariSpec hyperspectral camera attached to an epifluorescent microscope. The images produced by the system record the reflectance and absorption features of endospores based on the structure of the outer coat. Analysis of the HIM data was performed using accepted image and spectral processing routines. Where peroxide was the sporicide, changes in the outer endospore coat contributed to structurally significant visible and near infrared signature differences between live‐viable versus dead, non‐viable endospores. A statistical test for divergence, a method for scoring spectral structural diversity, also showed the difference between viable and non‐viable peroxide killed endospores to be statistically significant. These findings may lead to an improved optical procedure to rapidly identify viable and non‐viable endospores in situations of decontamination.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.     

Automatic quantification of mitochondrial fragmentation from two‐photon microscope images of mouse brain tissue

The morphology of mitochondria can inform about their functional state and, thus, about cell vitality. For example, fragmentation of the mitochondrial network is associated with many diseases. Recent advances in neuronal imaging have enabled the observation of mitochondria in live brains for long periods of time, enabling the study of their dynamics in animal models of diseases. To aid these studies, we developed an automatic method, based on supervised learning, for quantifying the degree of mitochondrial fragmentation in tissue images acquired via two‐photon microscopy from transgenic mice, which exclusively express Enhanced cyan fluorescent protein (ECFP) under Thy1 promoter, targeted to the mitochondrial matrix in subpopulations of neurons. We tested the method on images prior to and after cardiac arrest, and found it to be sensitive to significant changes in mitochondrial morphology because of the arrest. We conclude that the method is useful in detecting morphological abnormalities in mitochondria and, likely, in other subcellular structures as well.     

Condenser‐free contrast methods for transmitted‐light microscopy

Phase contrast microscopy allows the study of highly transparent yet detail‐rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser‐free yet highly effective method of obtaining phase contrast in transmitted‐light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light‐path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser‐free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser‐free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour‐contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser‐free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next‐generation transmitted‐light microscopy designs. The condenser‐free illumination method, using rings of independent or radially‐scanned emitters, may be exploited in future in other electromagnetic wavebands, including X‐rays or the infrared.     

Effects of metals cadmium and chromium alone and in combination on the liver and kidney tissue of male Spraque‐Dawley rats: An ultrastructural and electron‐energy‐loss spectroscopy investigation

Heavy metal pollution has increased in the last decades. Water sources are contaminated and human exposure is often long term exposure to variable amounts of different metals. In this study, male Sprague‐Dawley rats were exposed via oral gavage for 28 days to cadmium (Cd) and chromium (Cr), alone and in combination at concentrations 1000 times the human World Health Organization's acceptable water limits. Rat equivalent dosages were used. Blood markers of liver and kidney function were measured, changes to cellular morphology was determined with transmission electron microscopy and the intracellular metal localisation was determined with the electron energy‐loss spectroscopy and energy filtered transmission electron microscopy analysis. Both Cd and Cr caused changes to the nuclear and mitochondrial membranes and irregular chromatin condensation of hepatocytes. Cr exposure caused dilation of the rough endoplasmic reticulum (rER). The combination caused nuclear and mitochondrial membrane damage as well as irregular chromatin condensation. In the kidney tissue, Cd caused irregular chromatin condensation in the cells of the proximal convoluted tubule (PCT). Cr caused changes to the outer nuclear and mitochondrial membrane and chromatin structure. The combination group caused membrane damage, irregular chromatin condensation and rER changes in the PCT. All the metal groups showed damage to the endothelial cells and pedicles, but not to the mesangial cells. Cd and Cr bio‐accumulation was observed in the nucleus, mitochondria and rER of the liver and kidney and therefore are responsible for the cellular observed damage that can cause functional changes to the tissues and organs.     

Recent progress in freeze-fracturing of high-pressure frozen samples

Pancreatic tissue, bacteria and lipid vesicles were high‐pressure frozen and freeze‐fractured. In addition to the normal holder, a new type of high‐pressure freezing holder was used that is particularly suitable for suspensions. This holder can take up an EM grid that has been dipped in the suspension and clamped in between two low‐mass copper platelets, as used for propane‐jet freezing. Both the standard and the new suspension holder allowed us to make cryo‐fractures without visible ice crystal damage. High‐pressure frozen rat pancreas tissue samples were cryo‐fractured and cryo‐sectioned with a new type diamond knife in the microtome of a freeze‐etching device. The bulk fracture faces and blockfaces were investigated in the frozen‐hydrated state by use of a cryo‐stage in an in‐lens SEM. Additional structures can be made visible by controlled sublimation of ice (‘etching’), leading to a better understanding of the three‐dimensional organization of organelles, such as the endoplasmic reticulum. With this approach, relevant biological structures can be investigated with a few nanometre resolution in a near life‐like state, preventing the artefacts associated with conventional fixation techniques.     

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.     

Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation

We compare conventional infrared laser based three-photon excitation with a visible laser based two-photon excitation scheme for imaging the ultraviolet fluorophore serotonin in solution and in live cells. To obtain a signal level of 1000 photons per second per mM serotonin solution, we need a back aperture power of 5 mW at 550 nm (for two-photon excitation) and 33 mW at 740 nm (for three-photon excitation). The detectivity of serotonin (defined as the concentration of serotonin that yields a signal equivalent to three times the standard deviation of the signal obtained from the buffer alone) is 12 microM for two-photon, and 220 microM for three-photon excitation. Surprisingly, for live cell imaging of vesicular serotonin in serotonergic cells, three-photon excitation appears to provide better image contrast than two-photon excitation. The origin of this is traced to the concentration-dependent shift of the serotonin emission spectrum.     

Photo‐ and bio‐physical characterization of novel violet and near‐infrared lipophilic fluorophores for neuronal tracing

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion‐matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near‐infrared candidates. By combining two‐photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two‐photon action cross‐sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six‐colour neuronal tracing within the spinal cord and brain tissue.     

Ultrastructural effects of perfomic acid oxidation of epoxy-embedded tissue with and without early uranyl staining.

Epoxy-embedded blocks of glutaradehyde and OSO4 fixed adenohypophyseal tissue were immersed in performic acid, rinsed and sectioned for electron microscopy. Sections exhibited a general image intensity equivalent to control sections but their stainability with both uranyl acetate and lead citrate stains was increased. It was concluded that osmium was not removed by performic acid oxidation. Membranes of endoplasmic reticulum and the Golgi apparatus were not visible, and other components (acidophil granules, in particular) were markedly distored in shape after oxidation. Such changes, however, were not evident in specimens previously exposed to uranyl acetate during ethanol dehydration.     

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.     

Dimethyl‐2‐[(acridin‐9‐yl)methylidene]‐malonate as fluorescent probe for histochemical analysis

Fluorescent compounds have been widely used for biomolecule labeling in cytochemistry and histochemistry analysis. Here, it is described the optical properties of dimethyl 2‐[(acridin‐9‐yl)methylidene]‐malonate (LPSF/IP‐81), an acridine derivative. This compound was conjugated to Concanavalin A (Con A) lectin and applied as sugar probe in lectin histochemistry. Evaluation of luminescent properties showed that LPSF/IP‐81 is photoluminescent with excitation at 360 nm and emission at 428 nm. Con A hemagglutinating activity and LPSF/IP81 photoluminescence were unaltered after conjugation. Circular dichroism of Con A‐LPSF/IP81 conjugate showed the maintenance of the Con A structure. Lectin histochemistry with Con A‐LPSF/IP81 conjugate demonstrated different pattern recognition studying normal, fibroadenoma, and invasive ductal carcinoma of human breast. These findings indicate that LPSF/IP‐81 can be proposed as an alternative probe for histochemical analysis.     

High-resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens

Intracellular structures of rapidly frozen biological tissues were observed in 3-D under a low-temperature scanning electron microscope using a newly developed side-entry type cryo-holder. The present low-temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo-holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo-holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3-D configuration of lipid droplets which had been difficult to preserve by chemical fixation.     

THE EFFECTS OF EMISSION LIGHT PULSE WIDTH ON NON INVASIVE OPTICAL PLETHYSMOGRAPHIC DEVICES

Projecting pulsed light on biological tissue facilitates capturing in-depth optical plethysmographs to inspect micro-vascular blood flow. However, it remains unclear how the pulse width of light emission (t _pw) affects human tissue. In this study, alternating red and infrared light emitting diodes from a finger clip probe were set at 512 Hz. After conducting optical receiver sampling and digital filtering, the logarithmic ratio of the red and infrared light was used to examine their relationship to the pulse width. The pulse width varied from 70–1000 µs, yielding no observable effects on the ratios. These findings suggest that reducing the illumination time of optical components may reduce the amount of power consumed by the equipment and extend the lifetime of the power supply.     

Thermo‐oxidative stability of new‐generation base oils

Thermo‐oxidation of base oils is the primary cause of lubricant degradation and engine failure during use. Degradation is mainly due to high‐temperature oxidation and thermal decomposition near the piston ring zone, forming oxygenated compounds that polymerise to form high‐molecular‐weight insoluble deposits. New‐generation base oils are found to be more stable towards oxidation and deposit formation due to the absence of aromatics and polynaphthenes. However, compatibility with antioxidants and other additives is now of greater concern because of the poor solvency of these oils. With the increase in the purity of the oil, sometimes the oxidation performance is poor in comparison to group I oils, mainly due to the removal of sulphur compounds, which act as natural antioxidants. Thermal techniques, such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), are emerging as fast and accurate methods for determining the thermo‐oxidative stability of base oils and their additive blends, making it possible to measure the oxidation induction time, incipient oxidation temperature, and deposit‐forming tendency. The objective of this work is to evaluate the thermo‐oxidative stability of new‐generation group II/III base oils without antioxidant additives, using DSC and TGA. The kinetics of base oil oxidative degradation are studied using different heating rates. The data obtained from thermal techniques are correlated with the micro‐oxidation data obtained from the Penn State Micro‐Oxidation (PSMO) test. The response of a typical antioxidant additive, zinc dialkyldithiophosphate, towards oxidative degradation of base oils has also been studied.