A place for ultrastructural analysis of platelets in cerebral ischemic research

It is well known that estrogen is neuroprotective through various mechanisms which suggest that sex hormone levels, thrombotic mechanisms, and inflammatory processes are strongly interconnected in predicting the outcome and consequences of cerebral ischemia. Because platelet ultrastructure is altered in conditions like thrombosis and associated with stroke, the question arises whether ultrastructural analyses of platelet morphology may provide further insight into the role of estrogen during ischemic insult. In the current study, a hyperglycemic modification to the two‐vessel occlusion model for inducing experimental cerebral ischemia was employed, in order to correlate neural tissue integrity levels between three experimental groups to corresponding platelet ultrastructure so as to determine whether there is an association between cerebral ischemia and the presence of inflammatory or necrotic platelet ultrastructure. It is apparent in the results that under the influence of estrogen in cyclic or intact females, there is lesser neural tissue damage as well as a reduced degree of inflammation evident in platelet activation morphology when compared to males and acyclic or ovariectomized females. It is unmistakable that neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. It is therefore suggested that platelets may indeed be used successfully to follow the progression of events of cerebral ischemia and possibly assist in the assessment of treatment strategies and their effects on hemostasis. Microsc. Res. Tech. 76:795–802, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural characterization of surface‐induced platelet activation on artificial materials by transmission electron microscopy

Platelet adhesion is one of the most pivotal events of blood clotting for artificial surfaces. However, the mechanisms of surface‐induced platelet activation have not been fully been elucidated or visualized so far. In this study, we attempted to observe the internal structures and adhesion interfaces of human platelets attached to artificial surfaces by transmission electron microscopy (TEM) during the platelet activation process. We prepared observation samples by a conventional embedding method using EPON 812 resin. The sectioning was sliced perpendicular to the a‐platelet/material interface. Observation by TEM indicates that internal granules coalesce in the center of the platelet accompanied by pseudopodial growth in the early stage of platelet activation. Pseudopodia from a platelet attach to the material interface not along a plane but at a point. In addition, along with the process of platelet activation, the gap between the platelet membrane and the material surface at the interface disappeared and a‐platelet/material adhesion became much tighter. In the fully activated platelet stage, the platelet becomes thinner and tightly adheres to the substrate. As a result of comparative observation of an adherent platelet on polycarbonate (PC) and on amorphous carbon (a‐C:H), it was found that internal granules release was inhibited more remarkably on a‐C:H coating rather than on PC. Despite numerous technical difficulties in preparing sectional samples, such a study might prove the essential mechanism of biomaterial‐related thrombosis, and it might become possible to modify the surfaces of materials to minimize material‐related thrombosis. Microsc. Res. Tech. 76:342–349, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructure of activated mouse platelets: a qualitative scanning electron microscopy study

Platelets form an integral part of the coagulation process, and their ultrastructure can provide valuable information regarding diseases associated with hemostasis. During coagulation, platelets aggregate; this aggregation can be achieved in vitro, by adding thrombin to platelet-rich plasma. Previous research showed that human thrombin could be used successfully to activate mouse platelets. When conservative changes are included, the amino acid similarity between human and mouse thrombin is approximately 75%. In this qualitative study, we compare the ultrastructure of mouse platelet aggregates activated by human thrombin as well as two concentrations of mouse thrombin, using the scanning electron microscope. Results show that both human and mouse thrombin activate platelets to form aggregates with typical pseudopodia formation. Magnification up to 250,000x showed membrane morphology with the open canalicular system pores visible in both the mouse- and human-activated platelets. It is therefore concluded that mouse platelets can be successfully aggregated using either mouse or human thrombin.     

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high‐pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 77:453–457, 2014. © 2014 Wiley Periodicals, Inc.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.     

Comparing different preparation methods to study human fibrin fibers and platelets using TEM

For the study of cellular ultrastructure, the sample needs to be stabilized by fixation, with the ultimate aim to preserve the native tissue organization and to protect the tissue against later stages of preparation. Chemical and freezing fixation are most used, and chemical fixation employs agents that permeate tissues and cells by diffusion and covalently bind with their major biochemical constituents to fix them. Most widely used chemical fixatives are aldehydes, e.g., formaldehyde and glutaraldehyde, which are noncoagulating, crosslinking agents. Cryofixation methods for ultrastructural studies are also popular, and high-pressure freezing immobilizes all cell constituents and arrests biological activity by removing the thermal energy from the system. In the current research, we used platelet-rich plasma (PRP) to study expansive fibrin fibers and platelet ultrastructure to compare the two fixation techniques. We also used thrombin and calcium chloride as a clotting agent to determine the technique most suitable for the formation of extensive fibrin networks. Chemically fixated fibrin fibers were more compact and condensed and also showed a banding pattern on longitudinal sections. High-pressure frozen samples were more dispersed while platelets fixated showed better preserved cellular membranes and organelle structure. PRP coagulated by addition of CaCl(2) showed blood platelets that are noticeably more activated compared with PRP; however, with thrombin, a sharp ultrastructure was seen. We conclude that PRP mixed with thrombin, and freeze substituted, is the most suitable method for the study of extensive fibrin fibers as well as platelets.     

Molecular ultrastructure of the urothelial surface: Insights from a combination of various microscopic techniques

The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immuno‐TEM), and freeze‐fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno‐FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 77:896–901, 2014. © 2014 Wiley Periodicals, Inc.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

Geometric complexity is increased in in vitro activated platelets

This article investigates the use of computerized fractal analysis for objective characterization of the complexity of platelets in vitro stimulated by low level thrombin (0.02 U mL^?1), collected from healthy individuals and observed by means of transmission electron microscopy. Platelet boundaries were extracted by means of automatically image analysis. Local fractal dimension was evaluated by the box‐counting technique (measure of geometric complexity of the platelet outline). The results showed that the platelet boundary is fractal when observed by transmission electron microscopy and that, after an in vitro platelet activation test, the shape of platelets present increased geometric complexity in comparison to the no stimulated platelets (P < 0.001), with 100% correct classification. Computerized fractal analysis of platelet shape by transmission electron microscopy can provide accurate, quantitative, data to study platelet activation. The results may play important roles in the evaluation of the platelets status in pathological conditions, like as atherosclerosis and diabetes mellitus, where in in vivo activated platelets have been described. Microsc. Res. Tech. 78:485–488, 2015. © 2015 Wiley Periodicals, Inc.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

Geometric complexity identifies platelet activation in familial hypercholesterolemic patients

Familial hypercholesterolemia (FH), a genetic disease, is associated with a severe incidence of athero‐thrombotic events, related, also, to platelet hyperreactivity. A plethora of methods have been proposed to identify those activated circulating platelets, none of these has proved really effective. We need efficient methods to identify the circulating platelet status in order to follow the patients after therapeutic procedures. We propose the use of computerized fractal analysis for an objective characterization of the complexity of circulating platelet shapes observed by means of transmission electron microscopy in order to characterize the in vivo hyperactivated platelets of familial hypercholesterolemic patients, distinguishing them from the in vivo resting platelets of healthy individuals. Platelet boundaries were extracted by means of automatically image analysis. Geometric complexity (fractal dimension, D) by box counting was automatically calculated. The platelet boundary observed by electron microscopy is fractal, the shape of the circulating platelets is more complex in FH (n = 6) than healthy subjects (n = 5, P < 0.01), with 100% correct classification in selected individuals. In vitro activated platelets from healthy subjects show an analogous increase of D. The observed high D in the platelet boundary in FH originates from the in vivo platelet activation. Computerized fractal analysis of platelet shape observed by transmission electron microscopy can provide accurate, quantitative data to study platelet activation in familial hypercholesterolemia and after administration of drugs or other therapeutic procedures. Microsc. Res. Tech. 78:519–522, 2015. © 2015 Wiley Periodicals, Inc.     

Fine Structure of Bacterial Adhesion to the Epithelial Cell Membranes of the Filiform Papillae of Tongue and Palatine Mucosa of Rodents: A Morphometric,TEM, and HRSEM Study

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point‐dried, and coated with gold‐palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three‐dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods. Microsc. Res. Tech. 76:1226–1233, 2013. © 2013 Wiley Periodicals, Inc.     

Effect of streptolysin O on the microelasticity of human platelets analyzed by atomic force microscopy

Atomic force microscopy (AFM) has been shown to be a suitable tool to probe biophysical properties of cells and cell fragments. We analysed biophysical alterations of human platelets by AFM using streptolysin O (SLO) as a model for pore forming proteins. Permeabilization of platelet membrane by SLO was confirmed by transmission electron and confocal microscopy. Using force volume imaging combined with FIEL analysis we were able to show dynamically the increase in the elasticity of platelets during the pore formation by SLO and could correlate the viscoelasticity to the morphology of platelets. Stabilizing the actin cytoskeleton by phalloidin resulted in partial restoration of the elasticity indicating that loss of stability in platelets by SLO is mediated by alterations of both plasma membrane and cytoskeleton.     

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH‐6 clone 5). We directly observed the cultured cells on a pressure‐resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co‐localization of CD133 with F‐actin. An antibody against CD133 decreased cell migration. Methyl‐β‐cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration. Microsc. Res. Tech. 76:844–852, 2013. © 2013 Wiley Periodicals, Inc.     

Morphology and expression status investigations of specific surface markers on B‐cell chronic lymphocytic leukemia cells

The morphology of cells and expression status of specific surface markers [cluster of differentiation (CD)], such as CD5, CD19, CD20, CD38, and CD45, have long been considered as the essential indicators for the diagnosis and prognosis of B‐cell chronic lymphocytic leukemia (B‐CLL). Clinically, it is difficult to simultaneously obtain cell morphology and distribution of surface markers with flow cytometry, especially for some surrogate markers such as CD38. Here, as an alternative and complementary prognostic method, fluorescence microscopy and image processing method are introduced to directly visualize the cells from patients and to quantitatively determine the expression status of surface markers. In this study, the morphological parameters of B‐CLL cells were measured to establish the correlation between the cellular morphology and the surface marker expression. It was clear that the CD38+ and CD38? B‐CLL cells from the same CD38+ patients had hardly any size differences; however, an increase in perimeter was observed for CD38? patients. Moreover, the expression level of the receptors on the cell was independent of the cell size. There was no evidence showing that the expression intensities of CD19 and CD38 were related to each other for the CD38+ B‐CLL cells. On the same cells, CD5 was more selectively expressed on the cell membrane; however, the expression patterns suggested that the cell membrane of CD38? B‐CLL cells contained the least expression level of CD19. Microsc. Res. Tech. 76:1147–1153, 2013. © 2013 Wiley Periodicals, Inc.     

Microscopic,molecular, and biochemical investigations to characterize a benthic cyanoprokaryote Leptolyngbya strainfrom Egypt

Microscopic, molecular, and biochemical investigations were conducted to describe a benthic mat‐forming Leptolyngbya isolate collected from wastewater canal in Helwan area, Egypt. Microscopic examination revealed that the isolate was filamentous, nonheterocystous, with obvious granular surface ornamentation. Electron microscopy was used to reveal the isolate's ultrastructure. Cross walls were thick with uneven deposition. Thylakoids were convoluted and irregularly distributed. Granular content differed from one cell to the other probably due to their physiological stages/position within the filaments and/or their age. Nycridial cells were present. Highly refractile gas vesicle‐like structures were detected and their identity as gas vesicles was confirmed by amplifying the gene coding for the gas vesicle protein GvpA. The presence of gas vesicles in benthic microorganisms is intriguing, and it is possible that those vesicles serve as a floating and dispersal mechanism as they increase in filaments that are about to break and release vacuolated hormogonia. To further confirm the isolate's identity, molecular analysis using 16S rRNA gene was performed. The sequence showed only 94% similarity to Leptolyngbya badia and less than 92% to other leptolyngbya. The phylogenetic analyses showed the coclustering of this strain with other Leptolyngbya strain. The fatty acid composition, used as a chemotaxonomic marker, revealed the presence of a considerable amount of polyunsaturated acids. Nevertheless, saturated fatty acids represented the highest proportion of the total fatty. Surprisingly, fatty acids of relatively limited occurrence within oscillatorian cyanobacteria such as saturated myristic fatty acid and polyunsaturated fatty acid C16:3 were found. Microsc. Res. Tech. 76:249–257, 2013. © 2013 Wiley Periodicals, Inc.     

Morphology and lectin‐binding sites of pyloric caeca epithelium in normal and GnRH‐treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH‐treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH‐treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone‐treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH‐treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH‐sialidase‐PNA, KOH‐sialidase‐SBA as well as asialoglycans stained with HPA, SBA, GSA I‐B₄, LTA. N‐linked glycans were highlighted by Con A and KOH‐sialidase‐WGA. In GnRH‐treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH‐sialisase‐PNA in the apical region, KOH‐sialidase‐PNA, KOH‐sialidase‐DBA, HPA, SBA, KOH‐sialidase‐SBA and KOH‐sialidase‐WGA in apical granules, GSA I‐B₄ and LTA in the supranuclear region. GnRH‐treated specimens showed some columnar cells that stained with KOH‐sialidase‐WGA in the apical granules and with GSA I‐B₄ in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH‐sialidase‐DBA, SBA, GSA II. The latter three binding sites lacked in GnRH‐treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH‐treatment. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Visualization of single proteins from stripped native cell membranes: A protocol for high‐resolution atomic force microscopy

Atomic force microscopy (AFM) proved to be able to obtain high‐resolution three‐dimensional images of single‐membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high‐resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane. Microsc. Res. Tech. 76:723–732, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural studies on the natural leaf senescence of Cinnamomum camphora

The process of natural leaf senescence of Cinnamomum camphora (C. camphora)—a commercial tree in Asia, was investigated, focusing on changes in cellular ultrastructure, epicuticular wax, and stoma. The changes to mesophyll cells in a senescing leaf predominantly include degradation of the following cellular components: cytoplasm, the central vacuole, small vacuoles, and vesicles with a diameter smaller than 400 nm, which are involved in the degradation of chloroplasts. The sequence of change in epicuticular wax during leaf senescence was different from those in herbaceous plants by atomic force microscope and scanning electron microscopic analysis. Comparing with maturation leaves, senescing leaves develop a wider aperture in their stoma, which would delay the leaf senescence of C. camphora. SCANNING 35:336‐343, 2013. © 2013 Wiley Periodicals, Inc.