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Electron Microscopy
Abstract
Many staining techniques have been used in combination with light microscopy to study the distribution of lignin in wood (Brauns 1952). However, since the advent of electron microscopy, considerable efforts have been made to obtain a better understanding of lignin morphology in wood cell walls. One of the techniques in electron microscopy for detection of lignin involves the use of potassium permanganate (KMnO4) stains (Crocker 1921, Hepler et al. 1970, Bland et al. 1971, Parham 1974, Kutscha and Schwarzmann 1975, Saka et al. 1979). Although some doubts have been expressed as to its specificity for lignin (Hoffmann and Parameswaran 1976, Kishi et al. 1982), the KMnO4 staining technique provides many excellent details of the ultrastructural features of lignin in wood.
The principal techniques used to elucidate the complex chemical structure of lignin (NMR, IR, Raman, UV/vis, MS, etc.) are summarized. Special attention has been focused on the detection of characteristic functional groups and main types of links present in lignin.
The aim
of this study was to investigate the structural composition of lignin (LIPE®)
extracted from Eucalyptus grandis,
before and after exposure to the gastrointestinal tract of ruminants. For this
study lignin was isolated, purified and characterized using 1H and 13C
Nuclear Magnetic Resonance Spectroscopy, nitrobenzene oxidation, functional
groups analysis and gas chromatography with mass spectroscopy. To have a better
understanding of lignin morphology and of its ultra structure, electron microscopy
in wood cell wall was utilized. The lignin fecal samples showed similar 1H
NMR spectrums and nitrobenzene oxidation products compared with LIPE®.
The result of the microanalysis of LIPE® was similar to data shown in the
literature. The ultra structure of lignin was similar to that of Bamboo (hard
wood). The lignin isolated from feces of sheep was identical to the original
LIPE®, which means that this product can be used as an external marker in
protein digestibility in sheep.
It was pointed out by Chafe and Chauret (1974) that an isotropic layer and a protective layer,which characterize the layered structure of the secondary wall of xylem parenchyma in hardwoods, resemble each other in having primary wall nature except lignin deposition. In this study, chemical components of these two layers were examined using such assured of treatments on serial ultra thin sections as delignification, hemicelluloses removal, pectin extraction and total polysaccharides removal.
As the results, it can be said that both layers are rich in hemicelluloses and contain some pectic substances, scattered with some cellulose microfibrils among them, but poor in lignin, at first stage. They become lignin-rich after the deposition of inner secondary wall on them. Consequently, both layers may be the sam in their origin and their structures, so that it may be favorable not to separate them and call both the term “amorphous layer”.
In order to localize hemicelluloses in spruce holocellulose and pulp fibres at the ultrastructural level the aldehydic end groups of the polysaccharide chains were oxidized to carboxyl groups and these were contrasted with colloidal iron, silver or lead ions or KMnO4. It is shown that KMnO4is not a specific stain for lignin as generally assumed, rather it contrasts acidic groups as well. The highest concentration of hemicelluloses was found in the S1, the concentration decreasing towards its inner zones. The S1/S2 border region and the outermost parts of the S2 show a hemicellulose content higher than the inner S1. In the middle of the S2 the concentration is lower again and is constant except for a narrow region of higher content at the border to the tertiary layer (T). The T generally shows a hemicellulose concentration similar to that in the middle zone of S2. The dense contrast seen in the cell corners and compound middle lamella must be ascribed to hemicelluloses as well as to highly acidic pectins.
Ultraviolet absorbance and bromine uptake were measured on various morphological regions of birch wood. To facilitate precise comparison, observations were made on identical locations in successive cross sections. From the data, the concentration of lignin and the ratio of the guaiacyl to syringyl residues were determined. The results obtained were then compared with the previous findings from the use of UV microscopy alone.
A study of lignin model compounds indicated that the reactivity of non-condensed guaiacyl, condensed guaiacyl and syringyl nuclei towards bromine is different. In order to evaluate the overall reactivity of the guaiacyl and syringyl residues in lignin, two softwood and eight hardwood species, selected so as to cover a range of methoxyl groups per phenylpropane unit (MeO/C9) in their lignins, were treated with bromine and the extent of the bromination of the lignin was determined by elemental analysis. The results indicated that the overall reactivity of the syringyl lignin to bromine was 1.2 times higher than that of the guaiacyl lignin. This finding was in good agreement with the data from the brominated white birch tissue fractions of the middle lamella and secondary wall.
The distribution of lignin has been studied in tracheids and ray cells of normal and compression wood of tamarack [Larix laricina (Du Roi) K. Koch]. The three layers in the secondary wall of normal wood tracheids are lignified to approximately the same extent, and previous evidence that the S
3 layer should contain a higher proportion of lignin than the other regions has not been confirmed. The lignin follows closely the orientation of the cellulose microfibrils in all three layers. Compared to the tracheids, the ray cells contain a denser network of lignin in their secondary wall.
Only a small proportion of the total lignin in compression wood tracheids is present in the compound middle lamella. The thick S
1 layer is only slightly lignified; the orientation of the lignin in this region is that of the transversely oriented, lamellated microfibrils. The outer portion of S
2 consists largely of lignin but also contains lamellae of cellulose microfibrils which probably have the same helical orientation as the microfibrils in the inner part of S
2. The latter region, which contains the helical cavities, consists of lamellae of cellulose microfibrils which are uniformly encrusted with lignin. The ray cells in compression wood appear to be lignified to the same extent as in normal wood. Transverse sections of the cells reveal a lateral orientation of the lignin. The orientation of the cellulose microfibrils in the S
2 layer of the first-formed springwood tracheids of compression wood is the same as in the cells which are formed later. It is suggested that for ease of reference, the outer, lignin-rich layer in compression wood tracheids be referred to as the S
2(L) layer.
A sieving technique has been developed for the separation of middle-lamella fragments. The middle-lamella fraction as well as the whole wood and compression wood from Picea abies have been analysed by nitrobenzene oxidation and acidolysis in order to determine the content of p-hydroxyphenylpropane units in the middle-lamella lignin. These analyses revealed only traces of p-hydroxyphenylpropane units in the whole wood and in the middle-lamella fraction but considerable amounts were found in compression-wood lignin. This points to the fact that middle-lamella lignin is of guaiacyl nature and that earlier results reporting high proportions of p-hydroxyphenylpropane units in the middle lamella-lignin may be due to the inclusion of compression wood in the fraction studied. The acidolysis experiments further indicate that the middle-lamella lignin has fewer uncondensed ß-0-4 aryl ether structures than the whole wood lignin.
The loss of mass of wood and its polymeric constituents in transmission electron microscopy has been determined by measurement of the decrease in the continuum x-ray intensity for various doses of irradiation. It was found that for doses higher than 5 × 10−8 C/μm2, 42% of the original mass of the wood remained on the grid. The corresponding percentages for cellulose, xylan, and lignin were 32, 45, and 70, respectively. The significance of these results in the use of transmission electron microscopy for imaging and for quantitative microbeam analysis is discussed.
Ultraviolet absorption spectra were determined microscopically for the different morphological regions of fourteen hardwood species. The woods were chosen to represent a range of methoxyl to carbon nine ratio (MeO/C9) in their lignins. The various trends in absorbance and peak wavelength indicated that1.
Syringyl residues become increasingly predominant in the walls of the fiberes and ray cells as the MeO/C9 ratio of the lignin increases.
2.
The vessel walls and cell corner regions contain mostly guaiacyl residues except at high MeO/C9 ratios when the syringyl character of the vessel walls increases. If certain reasonable assumptions were made, it was possible to resolve the spectrum of the fibre wall of eastern cottonwood into spectra of the syringyl, guaiacyl and p-hydroxy benzoic acid components.
A new technique suitable for determination of lignin distribution in hardwoods has been developed. The technique is based on the mercurization of the aromatic nucleus of the lignin and a concomitant determination of the mercury content by the SEM- or TEM-EDXA technique. An incorporation of one mole mercury per mole lignin (C9-unit) was obtained for the birch lignin. The concentration of lignin was found to be about 3 times higher in the cell corners of the middle lamella than in the secondary wall of the birch fibers. The cell wall of the vessels and the ray cells were almost equal in lignin concentration and had about 1.5–1.6 times higher concentration than the secondary wall of the fibers. No specific mass loss of the organically bound mercury during the electron beam bombardment was found under the conditions used in this work.
The distribution of lignin in normal and tension wood of four hardwood species has been studied by examination in the electron microscope of the lignin skeletons remaining after removal of the polysaccharides with hydrofluoric acid. In normal wood fibers, the S1 had a higher lignin concentration than the S2 layer, which was not as highly lignified as in conifer tracheids. Vessels had a high concentration of lignin in both normal and tension wood, while the extent of lignification of the parenchyma was variable.
In tension wood fibers, the S1 and S2 layers were highly lignified. A thick, unlignified G-layer was often associated with an extremely thin S2 layer with a high concentration of lignin. In both normal and tension wood, the lignin had the same orientation as the cellulose micro-fibrils in the different cell wall layers. The results confirm the earlier conclusion that, in the species investigated, the same amount of lignin is present in gelatinous as in normal fibers. Evidently, the lignification mechanism operates normally in the non-gelatinous layers of the fibers, as well as in the vessels and in the parenchyma of tension wood.
A method has been developed for the determination of lignin distribution in the wood cell wall by ultraviolet microscopy. The method incorporates some important advances on previus applications of UV microscopy to the study of lignin distribution. Ultrathin cross-sections of wood are obtained by the sample preparation and sectioning techniques of electron microscopy. The specimens are examined in monochromatic ultraviolet light using quartz reflection optics. The microscope image is photographically recorded and the negative is subsequently subjected to densitometric analysis. Each stage of the analytical procedure has been critically assessed to determine its validity and limitations. The method is ideally suited to the study of the removal of lignin from the wood cell wall during cooking and possesses other important applications in wood technology.Es wurde ein Verfahren zur Bestimmung der Ligninverteilung in der Holzzellwand entwickelt, das sich der Ultraviolett-Mikroskopie bedient. Das beschriebene Verfahren beinhaltet einige wichtige Fortschritte genenber frheren Anwendungen der UV-Mikroskopie beim Studium der Ligninverteilung. Wie fr die Elektronenmikroskopie mssen durch Schnitttechnik und Probenprparation ultradnne Querschnitte der untersuchten Hlzer angefertigt werden. Die Proben werden dann in monochromatischem UV-Licht durchmustert unter Verwendung wendung eines Quarzreflektors. Das mikroskopische Bild wird photographisch festgehalten und das Negativ anschlieend einer densiometrischen Analyse unterworfen. Jede Stufe dieses Untersuchungsverfahrens wurde kritisch untersucht, um Gltigkeit und Grenzen des Verfahrens festzulegen. Das Verfahren eignet sich in idealer Weise zur Untersuchung der Herauslsung von Lignin aus der Holzzellwand whrend des Kochprozesses; es drfte darber hinaus andere wichtige Anwendungsmglichkeiten in der Holzforschung besitzen.
The lignin concentrations of the compound and cell corner middle lamella regions of the tracheid cell wall in radiata pine were determined relative to the S2 region using (i) interference microscopy and (ii) bromination in conjunction with energy dispersive X-ray microanalysis. Relative lignin concentration values for the cell corner middle lamella as determined by interference microscopy were 3.39 and 4.23 for the two specimens examined, while the corresponding values determined by bromination/EDXA using Br K X-rays were 1.95 and 1.21. Values for the compound middle lamella were 1.54 and 0.86 as determined by bromination/EDXA. Possible reasons for the poor agreement between the two techniques are discussed.
Elemental and functional group analyses were carried out on tissue fractions from the secondary wall and middle lamella of black spruce tracheids. The secondary wall lignin was found to contain 1.7 times as much methoxyl per C9 as the middle lamella lignin, indicating a substantial proportion of unmethylated para-hydroxyphenylpropane residues in the middle lamella. The content of carbonyl groups was at least three times larger in middle lamella lignin than in secondary wall lignin. The carboxyl content of the middle lamella was found to be about three times as large as that of the secondary wall. Elemental analyses show a higher carbon and lower oxygen content in the middle lamella than in the secondary wall.
In order to elucidate a previously reported discrepancy in the ratio of the lignin concentration in the middle lamella to that in the secondary wall as determined by ultraviolet (UV) microscopy and bromination combined with EDXA, the ultraviolet absorptivity of the lignin and the lignin reactivity towards bromination were compared for black spruce wood (Picea mariana Mill.). In addition, UV microscopy and EDXA techniques were applied to the determination of lignin distribution in the tracheids in order to establish the relationship between the two techniques. The results indicated that, although the ultraviolet absorptivity in different morphological regions is essentially the same, the secondary wall lignin was 1.70 times more reactive towards bromination than the middle lamella lignin. By applying the value of 1.70 as a correction to the EDXA results, the estimated lignin distribution by EDXA was in fairly good agreement with that from UV microscopy.
The lignin distribution between the middle lamella and the cell wall of spruce fibers has been determined by a new technique based on a mercurization of the lignin and a concomitant determination of mercury by the SEM-EDXA technique. The ratio of lignin in the middle lamella at the cell corners to the lignin in the secondary wall was 2.50.6 for latewood and 2.40.6 for earlywood. This gives a lignin content of 55–58% in the true middle lamella in the cell corners. The reactivity to mercuric acetate of different wood elements was determined in separate experiments. Fractions enriched in ray cells, middle lamella, and compression wood all reacted at the same rate as the whole wood; about one mole of mercury was incorporated per mole of lignin (C9-unit).
The distribution of lignin in black spruce has been determined quantitatively by the study of 0.5 μm transverse sections in a UV microscope. The average lignin concentration in the compound middle lamella was about twice that in the secondary wall. The lignin concentration of the middle lamella at the cell corners of adjacent tracheids was nearly four times that in the secondary wall but the volume of the secondary wall was much greater than the volume of the middle lamella. Thus, for earlywood, 72% of the total lignin was in the secondary wall leaving only 28% in the compound middle lamella and cell corner middle lamella regions. The corresponding values for latewood were 82% and 18% respectively. Use of oblique longitudinal sections of 0.1 μm thick permitted the resolution of the compound middle lamella. The lignin concentration in the true middle lamella was found to be equal to that in the cell corner middle lamella and the primary wall lignin content to be about twice that in the secondary wall.
The distribution of lignin in opposite wood has been studied by removing the polysaccharides with hydrofluoric acid and examining the resulting lignin skeletons in the electron microscope. The thick S3 layer was more highly lignified than the S1 and S2 layers in Abies balsamea, Picea rubens, Pinus resinosa, and Tsuga canadensis. In Picea rubens, but not in the other species, there was, adjacent to the S3 layer, a transition zone in S2 with a high concentration of lignin. The S3 layer varied considerably in thickness and was often buckled, especially in the latewood. The structure of the bordered pits was that observed in the original wood. The margo, the torus, and the initial pit border were all highly lignified.
The distribution of lignin in normal and compression wood of loblolly pine (Pinus taeda L.) has been studied by the technique of lignin skeletonizing. Hydrolysis of the wood carbohydrates with hydrofluoric acid left normal wood tracheids with a uniform distribution of lignin in the S1 and S2 cell wall layers. However, the S3 region of both earlywood and latewood tracheids consistently retained a dense network of unhydrolyzable material throughout, perhaps lignin.
Lignin content in compression wood averaged about 7% more than in normal wood and appears to be concentrated in the outer zone of the S2 layer. The inner S2 region, despite helical checking, is also heavily lignified. The S1 layer, although thicker than normal in compression wood tracheids, contains relatively little lignin.
Ray cells, at least in normal wood, appear to be lignified to the same extent, if not more so in certain cases, than the longitudinal tracheids. Other locations where lignin may be concentrated include initial pit border regions and the membranes of bordered pits.
A method has been developed for the determination of lignin distribution in wood cell walls by studying bromine concentrations in 0.5 m sections with scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDXA) technique. Evaluation of backscattering, absorption and fluorescence effects on quantitative assay of lignin distribution indicated that these effects may all be ignored, if relative comparisons are made. Thus, a relative ratio of the measured Br-L X-ray intensities directly provides bromine concentration ratio, from which the ratio of lignin concentrations in different morphological regions can be estimated. Additionally, the effect of electron beam bombardment on bromine was found to be negligible. Therefore, the SEM-EDXA technique provides quantitative information of lignin distribution with relatively high accuracy. The potential and limitations of this technique were also elucidated.
A Faraday cup has been constructed which is capable of accurately measuring beam‐current densities in the image plane of an electron microscope. This device has been employed to calibrate a solid‐state detector typical of those often used to measure such small electron intensities. The ability of the solid‐state detector to distinguish single electrons was found to be a sensitive function of the incident electron intensity. Important applications of this work include investigations of radiation damage in beam‐sensitive materials, e.g., biological specimens.
The exposure meter accessories for Siemens Elmiskop I and IA and Phillips EM 200 microscopes use the final screen as an electron collector. A robust and efficient Faraday cup is described, and this was used to calibrate the collection efficiency of these flat screens. The results in each case could be expressed as a linear function of beam voltage. A negligible fraction of the beam is scattered below the specimen level, so the beam current density at the specimen is obtained directly if the magnification is known. The Faraday cup has also been used in an AEI EM6G and in a Cambridge Instruments Mk 1 Stereoscan.
A low-viscosity embedding medium based on ERL-4206 is recommended for use in electron microscopy. The composition is: ERL-4206 (vinyl cyclohexene dioxide) 10 g, D.E.R. 736 (diglycidyl ether of polypropylene glycol) 6 g, NSA (nonenyl succinic anhydride) 26 g, and S-1 (dimethylaminoethanol or DMAE) 0.4 g. The medium is easily and rapidly prepared by dispensing the components, in turn by weight, into a single flask. The relatively low viscosity of the medium (60 cP) permits rapid mixing by shaking and swirling. The medium is infiltrated into specimens after the use of any one of several dehydrating fluids, such as ethanol, acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and tert.-butyl alcohol. It is compatible with each of these in all proportions. After infiltration the castings are polymerized at 70°C in 8 hours. Longer curing does not adversely affect the physical properties of the castings. Curing time can be reduced by increasing the temperature or the accelerator, S-1, or both; and the hardness of the castings is controlled by changes in the D.E.R. 736 flexibilizer. The medium has a long pot life of several days and infiltrates readily because of its low viscosity. The castings have good trimming and sectioning qualities. The embedding matrix of the sections is very resistant to oxidation by KMnO4 and Ba(MnO4)2, compared with resins containing NADIC methyl anhydride. Sections are tough under the electron beam and may be used without a supporting membrane on the grids. The background plastic in the sections shows no perceptible substructure at magnifications commonly used for biological materials. The medium has been used successfully with a wide range of specimens, including endosperms with a high lipid content, tissues with hard, lignified cell walls, and highly vacuolated parenchymatous tissues of ripe fruits.
The distribution of lignin in vessel walls after treatments on ultrathin sections
- K Kishi
- H Harada
- H Saiki
Calculation of lignin concentration and porosity of cell wall regions by interference microscopy
- J B Boutelje
- JB Boutelje
Lignin distribution in soda-oxygen and kraft fibers as determined by conventional electron microscopy
- S Saka
- Rj Thomas
- Js Gratzl
Tracheidal and parenchymatous cells in Picea abies (Karst.) pulpwood and their behavior in sulphite pulping
- E Back
The chemistry of lignin
- F E Brauns
- FE Brauns
Kvantitativ kemisk analys av cellväggens delar i ved-och cellulosafibrer med användning av interferensmikroskopi
- P W Lange
- A Kjaer
- PW Lange
Lignin distribution in Douglas-fir and loblolly pine as determined by energy dispersive X-ray analysis
- S Saka
- Rj Thomas
- Js Gratzl