Out-of-plane orientation of cellulose elementary fibrils on spruce tracheid wall based on imaging with high-resolution transmission electron microscopy

Main conclusion

A 3D model of the tracheid wall has been proposed based on high-resolution cryo-TEM where, in contrast to the current understanding, the cellulose elementary fibrils protrude from the cell wall plane. The ultrastructure of the tracheid walls of Picea abies was examined through imaging of ultrathin radial, tangential and transverse sections of wood by transmission electron microscopy and with digital image processing. It was found that the elementary fibrils (EFs) of cellulose were rarely deposited in the plane of the concentric cell wall layers, in contrast to the current understanding. In addition to the adopted concept of longitudinal fibril angle, EFs protruded from the cell wall plane in varying angles depending on the layer. Moreover, the out-of-plane fibril angle varied between radial and tangential walls. In the tangential S₂ layers, EFs were always out-of-plane whereas planar orientation was typical for the S₂ layer in radial walls. The pattern of protruding EFs was evident in almost all axial and transverse images of the S₁ layer. Similar out-of-plane orientation was found in the transverse sections of the S₃ layer. A new model of the tracheid wall with EF orientation is presented as a summary of this study. The outcome of this study will enhance our understanding of the elementary fibril orientation in the tracheid wall.     

Anatomy of the vessel network within and between tree rings of Fraxinus lanuginosa (Oleaceae)

The three-dimensional (3-D) arrangement of vessels and the vessel-to-vessel connections in the secondary xylem of the stem of the ring-porous hardwood tree Fraxinus lanuginosa were studied in series of thick transverse sections with epifluorescence microscope and confocal laser scanning microscope. Vessels were traced in sequential sections, and vessel networks were reconstructed in two segments of wood with dimensions of 2 × 1.4 × 21.2 mm(3) and 2 × 1.4 × 5.8 mm(3) (tangential × radial × axial). The arrangement of vessels and intervessel pits were visualized by scanning electron microscopy in low-density polyethylene microcasts and on exposed tangential faces of growth-ring boundaries. The vessels deviated from the stem axis in the tangential direction and, to a lesser extent, in the radial direction. Some neighboring vessels were twisted around each other. Vessels that appeared solitary in single sections were found to be sequentially contiguous with a number of other vessels, forming networks that extended in the tangential direction and across growth-ring boundaries. In the 21.2-mm wood block, all earlywood vessels at the growth-ring boundary made contact with latewood vessels in the previous tree ring. Within a growth ring however, only a single contact was observed between individual earlywood and latewood vessels. Densely arranged intervessel pits were characteristic in the regions where adjacent vessels made contact with each other. Such bordered pits were abundant in the tangential walls of vessel elements adjacent to growth-ring boundaries. Therefore, bordered pits appear to provide the pathway for the radial transport of water via the vessel network across growth-ring borders. Fiber-tracheids, observed as terminal cells in the tree rings, might also contribute to the apoplastic transfer of water across ring borders.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture

A thin section culture system for rapid regeneration of the monopodial orchid hybrid Aranda Deborah has been developed. Thin sections (0.6–0.7mm thick) obtained by transverse sectioning of a single shoot tip (6–7mm), when cultured in Vacin and Went medium enriched with coconut water (20% v/v), produced an average 13.6 protocorm-like bodies (PLB) after 45 days, compared to 2.7 PLB formed by a single 6–7 mm long shoot tip under same culture condition. Addition of -naphthaleneacetic acid to Vacin and Went medium enriched with coconut water further increased PLB production by thin sections. PLB developed into plantlets on solid Vacin and Went medium containing 10% (v/v) coconut water and 0.5 g l^–1 activated charcoal. With this procedure, more than 80,000 plantlets could be produced from thin sections obtained from a single shoot tip in a year as compared to nearly 11,000 plantlets produced by the conventional shoot tip method.Abbreviations BA 6-benzyladenine - CD callus development - CW coconut water - KC Knudson C medium - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PLB protocorm-like body - TS thin section - VW Vacin and Went medium     

A novel tight cylindrical mold for epoxy resin embedding allows enhanced microscopic analysis of microcores extracted from woody plants

A novel mold was devised to embed microcores extracted from stems of trees in epoxy resin, which has been widely used for optical and electron microscopic analysis of xylem formation. The embedding mold of a tight cylindrical shaped tube was designed to avoid displacement of microcores from the right position during the process of resin embedding. Microcores of a ring-porous hardwood species, Quercus crispula, with higher wood density and much larger differentiating vessel elements laid down on the boundary between the current xylem and the previous one, which generally cause difficulty in thin sectioning and breaks in sections, respectively, were embedded in the cylindrical molds full of epoxy resin. Locations of the three principal planes of wood anatomy could be determined in cylindrical resin-embedded microcores as follows: the transverse plane could be found on their side of cylinder, the radial one was vertical to the transverse, and the tangential ones were their circular ends of cylinder. The present embedding mold, therefore, can provide all three principal sections for microscopic wood anatomy from the side or ends of the same cylindrical microcore in principle. To confirm the usefulness of the resin-embedded microcores, we examined the differentiation of vessel elements during the period of earlywood formation on their transverse sections under microscopes, consequently could observe cell division in the cambial zone and sequential stages of vessel element differentiation, including cell expansion and deposition of the secondary cell wall. The present embedding mold for epoxy resin is simple but highly useful and innovative for a wide range of applications of microcores in microscopy for studies on tree-ring formation.     

Introduction and verification of a novel method for measuring wood fiber length using a single cross section in Acacia mangium

A novel method for calculating the wood fiber length using a single cross section was devised and verified in Acacia mangium. This method is based on the ratio of cell tips to total cell number in a cross section related to the wood fiber. The fiber length was calculated using the single cross-section method and was compared with the measurements obtained using the conventional maceration method and the serial cross-section method. There was no significant difference among the three methods.     

Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.     

Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction

By microinjecting rhodamine-conjugated pig brain tubulin into living pea stem epidermal cells it has been possible to follow cortical microtubules beneath the outer tangential wall (OTW) as they re-orientate from a transverse to a longitudinal alignment. Earlier immunofluorescence studies on fixed material have shown that parallel cortical microtubules circumnavigate the cell forming apparently continuous arrays which are transverse, oblique or longitudinal to the cell's long axis. If the array re-orientates as a whole then microtubules along the radial walls would be expected to share the alignment of those on the tangential walls. There are, however, reports that microtubules beneath the outer tangential wall have a different orientation from microtubules at the radial cell walls, raising important questions about the construction and behaviour of the array. Using computer-rotated stacks of optical sections collected by confocal scanning laser microscopy it has been possible to display the microtubules along radial as well as tangential walls of the same microinjected cells. These observations demonstrate for living epidermal cells that when microtubules are aligned longitudinally at the outer epidermal wall they remain oblique or transverse at the radial walls. The array may not therefore re-orientate as a whole but seems to undergo re-organization on only one cell face. However, despite the differing angles between the OTW and radial walls microtubules still form patterns which at the level of the confocal microscope are continuous from one cell face to another, around the cell.
It is concluded that some organizing principle attempts to establish overall organization at the cellular level but that this can be perturbed by local re-organization of dynamic microtubules in subcellular domains. This study emphasizes the importance of the outer epidermal wall and its associated cytoskeleton in initiating changes in the direction of cell expansion.     

A novel insight into the structure of amphivasal secondary bundles on the example of Dracaena draco L. stem

Key message

Pattern of tracheids found along the bundles extends understanding of their cross - sectional anatomy and sheds a new light on the issue of radial transport in monocotyledons with secondary growth.

Abstract

Secondary growth of Dracaena draco L. stem is connected with the formation of amphivasal vascular bundles in which a centrally located phloem is surrounded by a ring of xylem cells (tracheids). However, as visible in a single transverse section, there is a tendency towards variation among the secondary bundles from such with a xylem ring to ones in which the tracheids do not completely surround the phloem, i.e., are separated by vascular parenchyma cells. We aimed to elucidate the cross-sectional anatomy of amphivasal secondary bundles using the method of serial sectioning (with sections 3 μm thick), which allowed us to follow very precisely the bundle structure along its length. The analysis revealed that the xylem arrangement in these bundles depends on the position of a section in the bundle path. Each amphivasal bundle is composed of sectors where tracheids form a ring, as well as of such where tracheids are separated by vascular parenchyma cells. We hypothesize that this structure of amphivasal vascular bundles facilitates radial transport of assimilates to the sink tissues. The result of the anatomical analysis is discussed in a physiological context.     

17年生邓恩桉两个种源木材密度与干缩性研究

通过对邓恩桉两个种源木材基本密度和在湿、气、全干状态下的径、弦向尺寸的测定,研究了不同树干位置的基本密度和干缩率的变异规律,并运用加工工艺中减小木材皱缩和开裂措施(蒸汽调湿处理方法)探索皱缩开裂缺陷的恢复性能。结果表明:1)邓恩桉种源100(0.5265 g/cm³)的木材基本密度小于种源98(0.5360 g/cm³);2)木材基本密度在树干纵向呈波浪式变化,在横向边材显著大于心材;3)木材干缩率在纵向随高度的增加而减小,在横向边材大于心材,且弦向干缩率均为径向的1~3倍;4)经喷蒸调湿处理后,木材开裂程度减轻,且径向和弦向皱缩恢复率分别为0.4%、3.3%;5)木材基本密度与气干弦向干缩率存在极显著负相关(r=-0.313),并建立了气干干缩率与全干干缩率的回归模型。     

Microtubule organization in root cortical cells ofHyacinthus orientalis

Summary Overall cellular arrangement of cortical microtubules (MTs) is studied by reconstruction of MT images on serial thin sections. The mature root cortex ofHyacinthus orientalis L. cv. Delft blue is composed of elongate, highly vacuolate nondividing parenchyma cells. In longitudinal sections in these cells, MTs generally form parallel arrays at oblique angles to longitudinal cell axes. These MTs extend towards the transverse face of the cell where they appear in localized parallel arrays as well as in crisscross patterns. Repeated observations of oblique parallel arrays of MTs along the length of the cell and the continuity of MT bundles in serial sections suggest that MTs form a single helix in the cell. MTs in neighboring cells appear in sections either as parallel or as herringbone patterns, suggesting that the MT helices in these cells may spiral in the same or the opposite directions.Abbreviations MT Microtubule - MF microfibil - EM electron microscopy     

Anatomical features that facilitate radial flow across growth rings and from xylem to cambium in Cryptomeria japonica

Background and Aims

Although the lateral movement of water and gas in tree stems is an important issue for understanding tree physiology, as well as for the development of wood preservation technologies, little is known about the vascular pathways for radial flow. The aim of the current study was to understand the occurrence and the structure of anatomical features of sugi (Cryptomeria japonica) wood including the tracheid networks, and area fractions of intertracheary pits, tangential walls of ray cells and radial intercellular spaces that may be related to the radial permeability (conductivity) of the xylem.

Methods

Wood structure was investigated by light microscopy and scanning electron microscopy of traditional wood anatomical preparations and by a new method of exposed tangential faces of growth-ring boundaries.

Key Results

Radial wall pitting and radial grain in earlywood and tangential wall pitting in latewood provide a direct connection between subsequent tangential layers of tracheids. Bordered pit pairs occur frequently between earlywood and latewood tracheids on both sides of a growth-ring boundary. In the tangential face of the xylem at the interface with the cambium, the area fraction of intertracheary pit membranes is similar to that of rays (2·8 % and 2·9 %, respectively). The intercellular spaces of rays are continuous across growth-ring boundaries. In the samples, the mean cross-sectional area of individual radial intercellular spaces was 1·2 µm² and their total volume was 0·06 % of that of the xylem and 2·07 % of the volume of rays.

Conclusions

A tracheid network can provide lateral apoplastic transport of substances in the secondary xylem of sugi. The intertracheid pits in growth-ring boundaries can be considered an important pathway, distinct from that of the rays, for transport of water across growth rings and from xylem to cambium.Key words: Cryptomeria japonica, bordered pit, intercellular spaces, lateral transport, tracheid network, water conduction, xylem permeability     

Irregular helicoids in leech cocoon membranes

Helicoids in the cocoon membrane of leeches Theromyzon tessulatum and Erpobdella punctata comprise a twisted superposition of layers, each containing a variable number of planes formed by unidirectional fibrils. Straight fibrils intersecting at different angles were displayed in tangential sections through the cocoon wall of each species. When the sectioning angle was below a certain value (i.e., the critical angle), bow-shaped lines apparent in oblique sections were replaced by a succession of layers containing straight fibrils, permitting a direct measurement of step-angle change between successive layers in a helicoid. By this methodology, we determined that no regularities exist in the succession of step-angles or in layer thicknesses within the cocoon membranes, but that the distribution of step-angles between layers was unique for each cocoon type.     

On the Lamellar Structure of the Tracheid Cell Wall

Abstract: It is clear that cross sections of wood cells show a lamellar structure. This paper investigates the orientation of this lamellar structure of spruce (Picea abies) tracheids using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cross sections of spruce wood were produced through fracturing in longitudinal bending and tensile testing. When investigated with SEM, the fracture surfaces show a structure of mostly larger radial lamellae, in the order of 30 - 100 nm, i.e., agglomerations of a few cellulose aggregates. Thin transverse sections of the fracture zones investigated with atomic force microscopy show concentric lamellae with a width in the order of a single cellulose aggregate, i.e., 15 - 25 nm. No structural connection to the splinters in the radial direction can be seen. It is suggested that the radial lamellar structure is a consequence of the energy released during fracturing of the wood samples and that the undistorted wood has a concentric lamellar structure on a smaller structural level.     

Fine structural observations on the epidermis

Summary The organization of the wall of epidermal cells in the petiole of species of Apium, Eryngium, Rumex, and Abutilon as well as that of the epidermis of Avena coleoptile has been investigated. The outer and inner tangential walls consist of layers in which the cellulose microfibrils are oriented alternately parallel or transverse to the longitudinal cell axis. This organization resembles that previously described for collenchyma cell walls (Wardrop, 1969; Chafe, 1970). On the radial (anticlinal) walls the orientation of the microfibrils is transverse and these appear continuous with the layers of transverse orientation of the outer and inner tangential walls. Variation in thickness of the outer tangential, and radial, and inner tangential walls appears to result from the variation in thickness of those layers in which the microfibrils have a longitudinal orientation. The extent to which these observations can interpreted in terms of some type of modified multi-net growth is discussed.     

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining

Background

For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.

Results

Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.

Conclusions

A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.     

Three-dimensional stereotactic anatomy of the human trigeminal nerve nuclear complex

The trigeminal nuclear complex and its spinal tract extend throughout the greater part of the brain-stem and at medullary levels form the target site for producing stereotactic lesions. This paper describes a method for three-dimensional drawings of this nuclear complex. A stereotactic atlas of the human brain-stem and cerebellar nuclei has formed the data base. The two-dimensional composite transverse sections, at 1-mm intervals have been digitized using an X-Y coordinate plotting microscope. Computer programs have been written to generate drawings of a single transverse hemisection as well as regeneration of the opposite hemibrain-stem section. Specific programs were used to reconstruct serial transverse section outlines and incorporate the trigeminal nuclear complex with and without hidden line removal techniques and colour graphic display facilities. Rotation about the x, y and z axes was possible and permits any view of the reconstructed specimen to be computer-generated. A further program for reconstructing structures as stereopairs is presented.     

X-ray microdiffraction reveals the orientation of cellulose microfibrils and the size of cellulose crystallites in single Norway spruce tracheids

The microfibril angle (MFA) distribution and the size of cellulose crystallites in isolated double cell walls of Norway spruce (Picea abies [L.] Karst.) tracheids were determined by synchrotron X-ray microdiffraction using the reflections 200 and 004. Samples were 25 μm thick longitudinal sections of earlywood from annual rings 6–18 of several stems. The asymmetric MFA distributions extended from ?20° to 90°. The mean MFA of tangential cell walls decreased from an average of 24° into 19° from the pith to the bark. The mode of the MFA distribution was about 10° smaller than the mean MFA. The standard deviation of the MFA distribution varied between 18° and 25°. The mean MFA and the mode of the MFA distribution were larger in radial than in tangential cell walls. MFA distributions of mature wood samples exhibited a separate small peak at around 90°. The average width and length of cellulose crystallites varied between 28.9–30.9 Å and 192–284 Å, respectively. Both increased slightly as a function of annual ring number from the pith up to the 15th annual ring. An irrigation–fertilisation treatment of some of the stems resulted in longer cellulose crystallites compared to the untreated stems.     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.