Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.     

Quality assurance and authentication of herbal drug (Argyrolobium roseum and Viola stocksii) through comparative light and scanning electron microscopy

The quality assurance and authentication of crude herbal drugs play important role in the effective therapeutic effect of herbal drug and their products. There are many reported problems in quality assurance of herbal crude drugs concerning to their correct identification. The present study was designed with the aim to document the authentication and quality assurance of the herbal crude drugs (Argyrolobium roseum and Viola stocksii) thorough light microscopy (LM) and scanning electron microscopy (SEM). The detailed foliar anatomical studies showed polygonal epidermal cells having anticlinal walls in Argyrolobium roseum while rounded epidermal cells were observed in Viola stocksii. The anomocytic stomata type was observed in Argrolobium roseum while actinocytic was noticed in Viola stocksii. The pollen of studied species appeared as tricolporate showing reticulate exine sculpturing in Argrolobium roseum while fine perforations were recorded in Viola stocksii. Furthermore, quantitative analysis of phenols, flavonoids, and antioxidant activity showed high flavonoid and phenol content in Argyrolobium roseum as compared with Viola stocksii. It was observed that Argyrolobium roseum was discriminated from the Viola stocksii based on the leaf and pollen micromorphological traits by using LM and SEM techniques. It was concluded that LM and SEM techniques were found useful for the quality assurance of botanicals and their authentication.     

Detailed macro and micromorphology of grasses caryopsis using scanning electron and light microscopy

Micromorphology of 15 grass caryopsis was investigated through scanning electron microscopy for its implications in the identification of grasses. Both macro and micromorphological characters especially caryopsis shape and surface morphology were found significant in the taxonomic diagnosis. Shape morphology of caryopsis varied among the species and lanceolate, fusiform, narrow ellipsoidal, ovate, elliptical, plano-convex, fusiform, linear ovate, broadly elliptical, broadly pllipsoidal, plano-convex, oblique ovate, and subglobose shapes were recorded. Digitaria nodosa (3.31 × 0.7 mm) and Echinochloa crus-galli (3.26 × 1.44 mm) had the largest caryopsis size, while Acrachne racemosa (0.76 × 0.54 mm) and Eragrostis minor (0.62 × 0.38 mm) had the smallest caryopsis size. Macro and micromorphological characters were useful for the diagnosis of genus and species. Utilizing both qualitative and quantitative character taxonomic key at generic and species level were made.     

Pollen morphology and its taxonomic potential in some selected taxa of Caesalpiniaceae observed under light microscopy and scanning electron microscopy

Pollen micromorphology is not only used to check the functional and structural evolution in plants but also to solve the taxonomic problem related to the classification of plants. Therefore, keeping in view the significance of pollen traits, selected taxa of the subfamily Caesalpiniaceae was collected from different geographical regions of Pakistan. The species were then analyzed under both light microscopy and scanning electron microscopy techniques to investigate the importance of micromorphological characters of pollen in the identification and classification of species. Great variation was recorded in equatorial shape, surface ornamentation, tectum, polar diameter, equatorial diameter, and exine thickness. However, little variation was observed in pollen type, polar shape, and fertility of pollen. The equatorial shape of five types was observed: prolate, prolate-spheroidal, spheroidal-subprolate, subspheroidal-prolate, and subspheroidal. Four types of surface ornamentation, psilate, granulate, clavate, and perforate, were recorded. Tectum of five types, intactate, reticulate regulate, medium reticulate, tactate, and striate, was observed. Sexine was thicker than nexine in all studied species. The largest polar diameter was observed in Caesalpinia pulcherrima 64.1 μm while the smallest in Parkinsonia aculeata 26.1 μm. The largest equatorial diameter was found in C. pulcherrima of 70.25 μm whereas the smallest in P. aculeata 27.57 μm. All the pollens analyzed were tricolporate. All studied species have a fertility ratio of more than 90%. A taxonomic key was developed to show the variation in pollen features and delimit species for the correct identification. In conclusion, the pollen traits were found useful to define species boundaries at various taxonomic ranks and will strengthen the taxonomy of this subfamily. Besides, this study also explored the palynological traits and their implication in the taxonomy of the subfamily Caesalpiniaceae.     

Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau,Western Himalayas

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A‐shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.     

Light microscopy and scanning electron microscopy: Implications for authentication of misidentified herbal drugs

Plant‐based drugs have reached remarkable acceptability as therapeutic remedy for various diseases due to the adverse effects of contemporary medicines. This increasing popularity of herbal drugs leads to a growing herbal market for the development of plant‐based drugs, nutraceuticals, pharmaceuticals and cosmeceuticals. Herbal drug adulteration is a complex problem which currently has undeniable consequences on health and nutrition. Ambiguities in nomenclature, misidentification and resemblance of colour and texture of the crude herbal drugs are the major causes of adulteration. Three different species commercially marketed under the same trade name Halion are Lepidium apetalum, Asparagus officinalis, and Lepidium didymum. The genuine source of Halion is Lepidium apetalum, which is authenticated by using basic and advanced taxonomic techniques. Morphology, anatomy and palynology of the misidentified sources were done using light and scanning electron microscopic techniques for authentication. This study may help to set microscopic techniques as a tool to achieve quality and standardization of the genuine source of the herbal drug. Phytochemical analysis and biological screening is needed for the further establishment of authenticity and quality of herbal drugs.     

Palynological characteristics of some selected species of family Cucurbitaceae (Juss.) using light and scanning electron microscopy techniques

The pollen morphology of 11 species distributed in seven genera belonging to family Cucurbitaceae has been studied using light microscopy (LM) as well as scanning electron microscopy. Pollen morphology of this family is diverse and is almost europalynous. Nearly all types of pollen belonging to different size class and shape class with a variety of exine sculpturing are found in the family. Pollen grains are usually triporate to tricolporate, prolate, oblate‐spheroidal, spheroidal, to suboblate, radially symmetrical, and isopolar. The common exine pattern is reticulate of various forms ranging from finely reticulate to coarsely reticulate with some species having echinate type of exine ornamentation, for example, Cucurbita pepo and Praecitrullus fistulosus. Most of the studied species are somewhat triangular with respect to equatorial outlook and circular to elliptic in polar outlook. The palynological characteristics of the members of the family are useful in identification of taxa at generic level mostly. Based on the present study it is not recommended to wholly solely depend upon the pollen morphology for the identification of taxa especially at specific levels because very minor differences regarding pollen characters exist among the species of the same genera.     

Systematic significance of pollen morphology and foliar epidermal anatomy of medicinal plants using SEM and LM techniques

Alternative medicinal source is a hot issue within medicine community now a days due to various side effects of allotropic drugs; most importantly the multidrug resistance phenomena. Medicinal plants provide an attractive and effective alternative to modern synthetic drugs as they contain different types of bioactive compounds, having great potential in treatment of various diseases. The present study deals with identification of micromorphological characteristics of 10 ethnomedicinally important plants of Northern Pakistan, that is, Aster himalaicus C. B. Clarke, Artemisia maritima (Berg.) Wellkomm, Isodon rugosus (Wall. ex Benth.), Myricaria squamosal Desv, Pedicularis groenlandica Retz. Polygonum aviculare L, Salvia himmelbauer E. Peter, Swertia kingie Hook. f., Tanacetum dolichophyllum (Kitam) Kitam, Verbascum epixanthinum Boiss. & Heldr. as potential source for pharmaceutical industry. The characters studied were shape and size of pollen, polar diameter and equatorial diameter, epidermal cell shape and size, stomata width and length, trichome shapes, and type. Most of the species had tricolporate pollen type but hexacolporate pollen was also observed in Isodon rugosus, Polygonum aviculare, Salvia himmelbauer. Different types of exine sculpturing observed were foveolate, scabrate, lophate, regular, steriate, and alveolate. Minimum equatorial diameter was noted in Tanacetum dolichophyllum (12.4 μm) and maximum in Myricaria squamosal (20.9 μm). Shape of epidermis cell ranged from rectangular and irregular, polygonal and pentagonal. Different types of stomata like anisocytic, actinocytic, paracytic, and diacytic were noticed. Variations in microscopic characters observed in the current study have great potential to be used as identification tools by the taxonomists for further studies.     

Implication of palynological techniques for the authentication of adulterated drugs traded with the same name in different herbal markets of district Lahore,Pakistan

Adulteration in traded medicinal plants is a significant issue nowadays and use of these adulterated medicinal plants can impose harmful impact to end user. However, this problem can be overcome by ensuring the identification of traded medicinal plants which are used in making different herbal medicines. In this regard, palynological markers are considered to be an important taxonomic tool in the identification of original medicinal plant from its adulterant. Hence this study attempted to provide particular reliable palynological markers for distinguishing selected medicinal plants from their adulterants, that is, Cinnamomum verum versus Canella winterana, Cinnamomum tamala versus Cinnamomum obtusifolium, Gymnema sylvestre versus Gymnema lactiferum, Artemisia maritima versus Artemisia absinthium, Achillea millefolium versus Adhatoda vasaka, Sphaeranthus indicus versus Sphaeranthus africanus, Averrhoa carambola versus Butea monosperma, and Morus nigra versus Morus alba. Results demonstrated great variations in multiple palynological characters between original medicinal plant and its adulterant such as in pollen size, shape, colpi length, exine, intine thickness, and fertility. In equatorial view, circular to spheroidal shape of pollen was found in A. millefolium while oblate shape was observed in A. vasaka. Similarly B. monosperma pollen was 34 μm, whereas pollen of its adulterant A. carambola was 21 μm. Moreover, colpi length of A. maritima was 11.8 μm, whereas 4.5 μm in A. absinthium. Hence it can be concluded that palynological characters are commendably helpful in identification of genuine medicinal plant from its adulterant.     

Systematics study through scanning electron microscopy; a tool for the authentication of herbal drug Mentha suaveolens Ehrh

In all over the world, herbal drugs are usually adulterated with similar species or varieties due to incorrect identification. Most of herbal products devoid purity and quality, therefore an attempt was carried out to identify plant species and authenticate its herbal drug products from Mentha suaveolens. Microscopy tools provide an excellent platform to identify plants at species level. In this study, microscopic and pharmacokinetic parameters of M. suaveolens were observed. Plant species were collected from high diverse areas of Northern Pakistan. Macro and micro‐morphology including palynology and anatomical features were analyzed to study M. suaveolens. Species characteristics were studied, while implementing microscopic techniques for the delimitation and identification of the species. Traditionally Mentha species are used to cure several diseases that is, digestive disorders, respiratory disorders. Micromorphology (stem, leaves, flowers structure, length etc.), palynology (shape, size of pollen etc.), and anatomical characters (types of stomata, epidermal cell shape, and trichomes) were studied. Micromorphology and anatomical characters were of great interest and significance to discuss the taxonomy of the species. Taxonomic characters were studied to characterize and authenticate the species. The aim of the present study is to observe in detail the taxonomic identification of the species in term of morphology, palynology, and foliar epidermal anatomy for the correct identification along with their medicinal uses in the area.     

Palynological investigation of some selected species of family Fabaceae from Pakistan,using light and scanning electron microscopy techniques

Pollen morphology of 11 species of family Fabaceae that is, Trifolium alexandrinum, Trifolium resupinatum, Arachis hypogaea, Lathyrus aphaca, Medicago lupulina, Vicia sativa, Lathyrus odoratus, Pongamia pinnata, Melilotus indicus, Medicago polymorpha, Medicago sativa from Pakistan has been investigated by light and scanning electron microscopy. Pollen were generally tricolporate, radially symmetrical, isopolar, elliptic in equatorial view and triangular in polar view under LM. Results showed that pollens were per‐prolate (T. alexandrinum), prolate (T. resupinatum, V. sativa, L. odoratus, Melilotus indicus, M. polymorpha, M. sativa) and sub‐prolate (A. hypogaea, L. aphaca, M. lupulina, P. pinnata). The larger polar/equatorial (P/E) ratio was found in T. alexandrinum (2.26 μm) and the smallest was found in M. lupulina (1.21 μm). The exine of T. resupinatum was 3.00 μm in thickness while others posses smaller exine thickness. The larger pore diameter was found in P. pinnata (16.01 μm) while others have smaller. The length of colpi was larger in Arachis hypogaea (32.24) while others posses smaller. Eight types of surface ornamentation (Psilate, faintly rugulate). Perforate and rugulate to verrucate have been observed under SEM. The pollens were europalynous type. Pollen morphology proved to be useful for the specific delimitation and serve as a tool for the identification and classification of taxa at specific and generic levels and can also be used as a key for the taxonomic features. Diversity in exine sculpture is helpful indicative characters for the isolation of closely related species. Hence, it is clear that both qualitative and quantitative characters of pollen can be useful for differentiating between taxa at specific level.     

Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover-glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

Comparative light microscopy,scanning electron microscopy and transmission electron microscopy of selected organic walled microfossils

Two simple techniques are described and illustrated. The first is for the study of one specimen by both light microscopy (LM) and scanning electron microscopy (SEM). The second is for the study of one selected specimen by LM, SEM and in ultrathin section by transmission electron microscopy (TEM). Although these techniques were developed for the comparative study of Precambrian organic walled microfossils (OWMs), they could be used for a wide range of other specimens.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Pollen morphological investigations of family Cactaceae and its taxonomic implication by light microscopy and scanning electron microscopy

The family Cactaceae is the diversified group of angiosperm plants whose pollen statistics has been used for taxonomic identification. In this article, we present the pollen morphology of eight species belong to seven taxonomically complex genera of Cactaceae including Astrophytum, Cylindropuntia, Echinocereus, Echinopsis, Mammillaria, Opuntia, and Thelocactus using light and scanning electron microscopy. The pollen grains were acetolyzed, measured, described, and electron photomicrographs were taken. Cactaceae can be characterized by presenting different palynomorphological features including pollen type, sculpturing, polar and equatorial diameter, aperture orientation, exine thickness, P/E ratio, and echini features. Four types of pollen shapes, that is, prolate spheroidal (three species), subprolate (two species), prolate (two species), and oblate spheroidal in Echinocereus reichenbachii were observed. The polar and equatorial diameter observed maximum in O. ficus indica 116.95 and 112.27 μm while minimum in M. compressa 38.42 and 21.05 μm. Pollen of two types, tricolpate in members of subfamily Cactioideae and pantoporate in the Opuntioideae were examined. The fertility percentage has been observed maximum in Opuntia macrocentra (83.84%) and minimum in Opuntia ficus‐indica (57.89%). Exine sculpturing showing great variations such as granulate, reticulate, granulate perforate and micro‐echinate foveolate ornamentation was examined only in Echinopsis eyriesii. A key to species, based on pollen micromorphological attributes, has been constructed for correct identification of complex cactus species.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.