Development of the reproductive system of Echinostoma paraensei in Mesocricetus auratus analyzed by light and confocal scanning laser microscopy

This study was performed to gain insight into the maturation of the reproductive system of Echinostoma paraensei worms grown in an early infection of Mesocricetus auratus. Hamsters were infected with 100 metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stained with hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanning laser microscopy. The average worm recovery was 43.7 per host. Images of the male and female reproductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, while the ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovary as a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs were already present during the first week of life. The two testes were seen as individual structures on 7 dpi while the cirrus sac and vitelline glands were in development. The ovary was connected to the uterus while the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at different stages of development, but with few bundles of functional spermatozoa in 10 day-old worms. On day 14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytes appeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraensei was functional on 14 dpi in the hamsters.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.     

Structural analysis of microparticles by confocal laser scanning microscopy

This study demonstrates the potential of conforcal laser scanning microscopy (CLSM) as a characterization tool for different types of microparticles. Microparticles were prepared by various methods including complex coacervation, spray drying, double emulsion solvent evaporation technique, and ionotropic gelation. Protein drugs and particle wall polymers were covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs were labeled by mixing with a fluorescent marker of similar solubility properties. As was demonstrated in several examples, CLSM allowed visualization of the polymeric particle wall composition and detection of heterogeneous polymer distribution or changes in polymer matrix composition under the influence of the drug. Furthermore, CLSM provides a method for three-dimensional reconstruction and image analysis of the microparticles by imaging several coplanar sections throughout the object. In conclusion, CLSM allows the inspection of internal particle structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the particle wall composition.     

Confocal laser scanning microscopy of fluorescently stained wood cells: a new method for three-dimensional imaging of xylem elements

Summary Xylem cells were fluorescently stained with periodic acid — Schiff reaction or with Schiffs reagent alone and studied by confocal laser scanning microscopy. Single images with extremely low depth of focus, series of optical sections, computed stereo scopic images and shadow casting images as well as x-z images are obtained. In contrast to scanning electron microscopy, not only are the surfaces imaged, but elements concealed by other structures can be visualized by this system. Quantitative data on cell depth are provided and differences in lignification are detected.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

基于高通量共聚焦激光扫描显微镜方法定量分析副溶血性弧菌生物被膜结构

【目的】副溶血性弧菌是水产品中常见的食源性致病菌,生物被膜的形成对副溶血性弧菌的环境生存和传播至关重要。这项工作的目的是评估临床和环境中分离出的44株副溶血性弧菌菌株形成的生物被膜的结构多样性。【方法】该研究基于共聚焦激光扫描显微镜的高通量方法,使用与高分辨率成像兼容的96孔微量滴定板,结合结构分析软件ISA-2来研究生物被膜形成和结构,分析22株食品与22株临床来源的副溶血性弧菌菌株形成的生物被膜结构参数(生物体积、平均厚度、粗糙系数)。【结果】CLSM图像显示,44株副溶血性弧菌菌株在培养48h后能够形成3D结构,进一步比较分析了临床来源菌株与环境来源菌株形成的生物被膜结构异同,发现临床菌株生物被膜的变异系数比环境菌株生物被膜的变异系数小,且同时携带tdh和trh两种毒力因子的菌株生物被膜变异性最小。凝聚层次聚类分析结果显示,副溶血性弧菌生物被膜可以分为致密且表面光滑(39%)、斑驳且表面粗糙(27%)、疏松且表面坑洼(34%),临床菌株易形成致密且表面光滑和斑驳且表面粗糙的生物被膜,而环境菌株易形成致密且表面光滑和疏松且表面坑洼的生物被膜。【结论】该研究深入了解了副溶血性弧菌生物...     

四唑盐减低法结合激光共聚焦显微镜定量分析表皮葡萄球菌生物被膜体外模型

目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。     

Confocal laser scanning microscopy of microtubule organizational changes in isolated generative cells of Allemanda neriifolia during mitosis

Summary Organizational changes in the microtubules of isolated generative cells of Allemanda neriifolia during mitosis were examined using anti--tubulin and confocal laser scanning microscopy. Due to an improved resolution and a lack of out-of-focus interference, the images of the mitotic cytoskeleton obtained using the confocal microscope are much clearer than those obtained using the non-confocal fluorescence systems. In the confocal microscope one can see clearly that the spindle-shaped interphase cells contain a cage-like cytoskeleton consisting of numerous longitudinally oriented microtubule bundles and some associated smaller bundles. At prophase, the shape of the cells invariably becomes spherical. The microtubule cytoskeleton inside the cells concomitantly changes into a less organized form — consisting of thick bundles, patches, and dots. This structural form is not very stable, and soon afterwards the cytoskeleton changes into a reticulate network. Then the nuclear envelope breaks down, and the microtubules become randomly dispersed throughout the cell. Afterwards, the microtubules reorganize themselves into a number of half-spindle-like structures, each possessing a microtubule-nucleating center. The locations of these centres mark out the positions of the presumptive spindle poles. Numerous microtubules radiate from these centres toward the opposite pole. At metaphase, the microtubules form a number of bipolar spindles. Each spindle has two half-spindles, and each half-spindle has a sharply focused microtubule centre at the pole region. From the centres, kinetochore and non-kinetochore microtubules radiate toward the opposite half-spindle. At anaphase A, sister chromatids separate, the cells elongate, and the kinetochore microtubules disappear; the non-kinetochore microtubules, however, remain, and a new array of microtubules, in the form of a cage, appears. The peripheral cage bundles and the non-kinetochore bundles coverge into a sharp point at the pole region. Later, at anaphase B the microtubule cytoskeleton undergoes reorganization giving rise to a new array of longitudinally oriented microtubule bundles in the cell centre and a cage-like cytoskeleton in the periphery. At telophase, some of the cells elongate further, but some become spherical. The microtubules in the central region of the elongated cell become partially disrupted due to the formation of a phragmoplast-junction-like structure in the mid-interzone region. The microtubule bundles at the periphery are spirally organized, and they appear not to be disrupted by the phragmoplast-like junction. The microtubules in the spherical telophase cells (unlike those seen in the elongated telophase cells) are arranged differently, and no phragmoplast-junction-like structure forms in the spherical cells. The structural and functional significances of some of these new features of the organization of the microtubule cytoskeleton as revealed by the confocal microscope are discussed.     

Valine uptake by the scleractinian coral Galaxea fascicularis: characterization and effect of light and nutritional status

The characteristics of valine uptake by isolated microcolonies of Galaxea fascicularis (Linnaeus 1758) were studied under various conditions including light, dark and feeding. The results demonstrated the presence of: (1) a linear component which might represent either a diffusional transport or a low-affinity carrier-mediated transport (apparent carrier affinity >250 mol·l^–1), and (2) a high-affinity active carrier-mediated transport (apparent carrier affinity about 5 mol·l^-1). The latter is mediated by two different systems: (i) a Na⁺-dependent carrier, stimulated by light and operative in both fed and unfed polyps, and (ii) a Na⁺-independent carrier, light insensitive and present only in unfed polyps. Competition experiments with other amino acids show that the Na⁺-dependent carrier is highly specific for neutral amino acids, as indicated by the high inhibition constants of basic and acidic amino acids. Our results suggest that the energy supplied by zooxanthellae photosynthates is necessary for the process of amino acid uptake, and that the Na⁺-dependent carrier responsible for valine uptake by G. fascicularis is similar to the B^0,+ system.Abbreviations AA amino acid(s) - AC/HC ratio autotrophic/heterotrophic carbon - ASW artificial sea water - DOM dissolved organic material - HPLC high performance liquid chromatography - K ₁ apparent inhibition constant - K _m apparent affinity of the carrier - SE standard error - V _max maximal rate of absorption     

Development of Schistosoma mansoni in the laboratory rat analyzed by light and confocal laser scanning microscopy

The kinetic of maturation (schistogram) of Schistosoma mansoni worms grown in laboratory rats was studied by light and confocal laser scanning microscopy. Infected rats with the BH strain were weekly euthanized 3-9 weeks pi. Recovered flukes stained with hydrochloric carmine were preserved as whole-mounts and analyzed by confocal and brightfield microscopy. Worms displayed varying degrees of maturation of the reproductive system at weeks 3-6. Male worms showed complete maturation of the reproductive system at week 6, while female worms completed their maturation at week 7. Males presented few tubercles in tegument in all weeks. Despite the presence of a developing embryo within the ootype, no uterine egg was found. The schistogram in rats follows a pattern similar to that observed in mice hosts.     

Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Observation of Protoplasts from the Conidia of Arthrinium Aureum by scanning electron microscopy

Protoplast formation from conidia of Arthrinium aureum was achieved with Lysing Enzyme L-2265 (Sigma Chemical) from Trichoderma harzianum. Scanning electron microscopy of conidia protoplasts showed the acquired spherical shape.     

Confocal laser scanning and electron-microscopic analyses of the relationship between VIP-like and GnRH-like-immunoreactive neurons in the lateral septal-preoptic area of the pigeon

The lateral septum and the preoptic area of birds comprise neurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP) and gonadotropin-releasing hormone (GnRH). By use of immunohistochemical single- and double-labeling techniques, we have investigated the distribution and the connections of these two types of peptidergic neurons in the lateral septal-preoptic area of the pigeon at both the light- and electron-microscopic levels. An accumulation of VIP-like-ir neurons, some of which are cerebrospinal fluid-contacting neurons, is found in the area adjacent to the ventromedial walls of the lateral ventricles in the lateral septum corresponding to the medial part of the lateral septal organ. VIP-like-ir terminals are scattered throughout the lateral septal-preoptic area, which also contains GnRH-like-ir cell bodies. The number of GnRH-like-ir cell bodies in the lateral septum is smaller than that of the VIP-like-ir neurons. GnRH-like-ir cells have a simple bipolar or multipolar shape and a beaded axon that emerges from the soma or one of the proximal dendrites. Confocal laser scanning microscopy has shown VIP-like-ir terminals in close apposition to GnRH-like-ir cell bodies in the lateral septal-preoptic area. Furthermore, the electron-microscopic double-immunolabeling has revealed synaptic contacts between VIP-like-ir axon terminals and GnRH-like-ir cell bodies or dendrites. These contacts, however, do not show synaptic specializations. The present results suggest that functional interactions take place between VIP and GnRH neurons in the lateral septal-preoptic area of the pigeon and that these interactions are involved in mediating photoperiodic responses. Received: 14 November 1997 / Accepted: 19 December 1997     

In vivo light-microscopic documentation for primary calcification processes in the hermatypic coral Stylophora pistillata

Skeletogenesis in the hermatypic coral Stylophora pistillata was studied by using the lateral skeleton preparative (LSP) assay, viz., a coral nubbin attached to a glass coverslip glued to the bottom of a Petri dish. Observations on tissue and skeletal growth were made by polarized microscopy and by using vital staining. The horizontal distal tissue edges developed thin transparent extensions of ectodermal and calicoblastic layers only. Four stages (I-IV) of skeletogenesis were observed at these edges, underneath the newly developed tissue. In stage I, a thin clear layer of coral tissue advanced 3–40 μm beyond the existing LSP peripheral zone, revealing no sign of spiculae deposition. At stage II, primary fusiform crystals (1 μm each) were deposited, forming a primary discontinuous skeletal front 5–30 μm away from the previously deposited skeleton. During stage III, needle-like crystals appeared, covering the primary fusiform crystals. Stage IV involved further lengthening of the needle-like crystals, a process that resulted in occlusion of the spaces between adjacent crystals. Calcification stages I-III developed within hours, whereas stage IV was completed in several days to weeks. Two basic skeletal structures, “scattered” and “laminar” skeletons, were formed, integrating the growth patterns of the needle-like crystals. High variation was recorded in the expression of the four calcification stages, either between different locations along a single LSP or between different preparations observed at the same diurnal time. All four skeletogenesis stages took place during both day and night periods, indicating that an intrinsic process controls S. pistillata calcification. This study was supported by the Israel Science Foundation (206/01 to J.E.), by the BARD, US-Israel Bi-National Agricultural Research and Development, by INCO-DEV project (REEFRES), and by CORALZOO, EC Collective Research project.     

Water droplets in intercellular spaces of barley leaves examined by low-temperature scanning electron microscopy

Low-temperature scanning electron microscopy was used to examine fracture faces in leaf blades taken from well-watered or drought-stressed barley (Hordeum vulgare L. cv. Mazurka) seedlings. The leaf blades were freeze-fixed while hydrated and were examined with or without gold-coating. There were droplets (with a smooth surface at the resolution achieved) on the surface of cell walls in leaf blades (0.91 g^-1 water content) from well-watered seedlings grown in an environment of 67% relative humidity. These were mainly on the vascular bundle sheath, the guard and subsidiary cells, and on some mesophyll cells around the substomatal cavity and between the stoma and vascular bundle. The droplets occurred, more abundantly, in the same places in seedlings from 100% relative humidity. They occurred on a few guard cells from wilting leaf blades (0.81 g·g^-1 water content) and were absent from severely drought-stressed leaf blades (0.15 g·g^-1 water content). The droplets sublimed at the same moment as both water which was in leaf cells and water which was allowed to condense (after freeze-fixation) on the wall surface. It is suggested that the droplets are aqueous. Their possible origin and importance is discussed.     

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.Abbreviations BSA Bovine serum albumin - CGRP calcitonin generelated peptide - DiA carbocyanine dye A - DiI carbocyanine dye I - dmnX dorsal motor nucleus of vagus - DMSO dimethylsulfoxide - ENK enkephalin - FITC fluorescin isothiocyanate - NADPH diaphorase nicotinamide adenine diphosphate - NPY neuropeptide Y - NTS nucleus tractus solitarii - PBS phosphate-buffered saline - VIP vasoactive intestinal peptide - WGA-HRP wheat-germ agglutinine-horseradish peroxidase     

Microfilament distribution in protonemata of the mossCeratodon

Summary Microfilaments were visualized in dark-grown protonemata of the mossCeratodon to assess their possible role in tip growth and gravitropism. The relative effectiveness of rhodamine phalloidin (with or without MBS) and of immunofluorescence (using the C4 antibody) was evaluated for actin localization in the same cell type. Using immunofluorescence, microfilaments were primarily in an axial orientation within the apical cell. However, a more complex network of microfilaments was observed using rhodamine phalloidin after MBS pretreatment, especially when viewed by confocal laser scanning microscopy. This method revealed a rich three dimensional network of fine microfilaments throughout the apical cell, including the extreme apex. Although there were numerous internal microfilaments, peripheral microfilaments were more abundant. No major redistribution of microfilaments was detected after gravistimulation. The combination of MBS, rhodamine phalloidin, and confocal laser scanning microscopy preserves and reveals microfilaments remarkably well and documents perhaps the most extensive F-actin network visualized to date in any tip-growing cell.Abbreviations BSA bovine serum albumin - CLSM confocal laser scanning microscopy - DIC differential interference contrast - DMSO dimethylsulfoxide - EGTA ethylene glycol bis-(-amino-ethylether) N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MEOH methanol - PBS phosphate buffered saline - PFA paraformaldehyde - PIPES piperazine-N,N-bis-2-ethanesulfonic acid - PMSF phenylmethyl sulfonyl fluoride - RP rhodamine phalloidin     

Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using spectral confocal laser scanning microscopy

In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.