Morphological and microscopic identification of three major medicinal Dendrobium species in Ta‐pieh Mountains area

Dendrobium is an important medicinal material in China. It has the effect of nourishing the stomach, nourishing yin and clearing heat. In China, there are many types of Dendrobium, and different Dendrobium species have different efficacy. The present study is aimed at distinguishing three major Dendrobium species from morphological and microscopic identification in Ta‐pieh mountains area. In this article, the roots, stems and leaves of Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme are used as materials to compare the differences of tissues of these three Dendrobium species by morphological indexes and microscopic identification of different Dendrobium. The stem morphology of these three Dendrobium species was significantly different except for stem internodes number and the middle part of the stem diameter by measuring the stems of Dendrobium. To ensure the safe use of Dendrobium, we built a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. huoshanense, D. officinale, and D. moniliforme. The microscopic results show that different types of Dendrobium exhibit different states that can be distinguished under normal light normal and fluorescence microscopy. This comparative study of morphology and microscopy contributes to the development of identification and quality evaluation of Dendrobium.     

Application of microscopy for discrimination of eight Swertia species utilized in the traditional Chinese medicine “Qingyedan”

Many Swertia species are utilized as a traditional medicine under the name “Qingyedan” in China, but are easily confused with one another. To distinguish eight Swertia species (S. mileensis, S. cincta, S. patens, S. punicea, S. delavayi, S. nervosa, S. macrosperma, and S. yunnanensis) and to ensure their safety and efficacy, the microscopic and macroscopic characteristics of the roots, stems, leaves, and flowers of them were examined. The results showed that microscopic and macroscopic features helpful for authentication of the eight species were the sinuosity of the anticlinal walls of epidermal cells and presence or absence of hairs on the leaf lamina; presence or absence of V‐shaped fibers and fibers with sinuous abaxial wall in the sepals; shape of epidermal cells and pattern of papillae on hairs on the margin of corolla nectary; distribution of stomata in leaf and sepal epidermises, stone cells in cortex and phloem of roots and in cortex and pith of stems, crystals in parenchymatous cells of mesophyll and stem, stomata size, stem diameter, and 4‐ or 5‐merous flowers, and so on. Two keys to the eight Swertia species based on macroscopic and microscopic characteristics are presented. The study indicates that microscopy and related techniques are convenient, practicable, and can be unambiguously applied for authentication of Swertia species. Microsc. Res. Tech. 76:296–310, 2013. © 2013 Wiley Periodicals, Inc.     

Authenticating and distinguishing the eight species of traditional Tibetan medicine “Meiduoluomi” by microscopic technique

Because of the morphological and macroscopic similarity, many species of Erigeron and Aster (Asteraceae) are confusable and usually used under the same name “Meiduoluomi” in traditional Tibetan medicine (TTM). To find an easy, quick, and reliable method to authenticate and distinguish the eight main medicinal plants of these species, the light microscope was used to reveal the morphoanatomic details. The fixed, sectioned, and stained plant materials and epidermis materials were studied by microscopic techniques. The results of the microscopic features are systematically described and illustrated, and comparison parameters are presented. Furthermore, a key to the eight species of “Meiduoluomi” was constructed. Microscopy can be unambiguously used to authenticate and distinguish the eight main species of TTM “Meiduoluomi.” Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Comparative authentication of three “snow lotus” herbs by macroscopic and microscopic features

“Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily‐confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.‐Bip, Saussurea laniceps Hand.‐Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech.1 77:631–641, 2014. © 2014 Wiley Periodicals, Inc.     

Identification of four Aconitum species used as “Caowu” in herbal markets by 3d reconstruction and microstructural comparison

Authentication is the first priority when evaluating the quality of Chinese herbal medicines, particularly highly toxic medicines. The most commonly used authentication methods are morphological identification and microscopic identification. Unfortunately, these methods could not effectively evaluate some herbs with complex interior structures, such as root of Aconitum species with a circular conical shape and an interior structure with successive changes. Defining the part that should be selected as the standard plays an essential role in accurate microscopic identification. In this study, we first present a visual 3D model of Aconitum carmichaeli Debx. constructed obtained from microscopic analysis of serial sections. Based on this model, we concluded that the point of largest root diameter should be used as the standard for comparison and identification. The interior structure at this point is reproducible and its shape and appearance can easily be used to distinguish among species. We also report details of the interior structures of parts not shown in the 3D model, such as stone cells and cortical thickness. To demonstrate the usefulness of the results from the 3D model, we have distinguished the microscopic structures, at their largest segments, of the other three Aconitum species used for local habitat species of Caowu. This work provides the basis for resolution of some debate regarding the microstructural differences among these species. Thus, we conclude that the 3D model composed of serial sections has enabled the selection of a standard cross‐section that will enable the accurate identification of Aconitum species in Chinese medicine. Microsc. Res. Tech. 78:425–432, 2015. © 2015 Wiley Periodicals, Inc.     

Microscopic study on the concretion of ceramics in the “Nanhai I” shipwreck of China,Southern Song Dynasty (1,127–1,279 A.D.)

“Nanhai I” shipwreck of China Southern Song Dynasty is the oldest and the most integrally preserved shipwreck in the world. The related conservation and archeological research have caught great attention of different experts all over the world. In this study, different types of concretion covered on the surface of the ceramics in “Nanhai I” shipwreck were analyzed by X‐ray diffractometer, micro‐Raman spectrometer, and scanning electron microscope equipped with energy dispersive spectroscopy. Based on the analyses, we found that the grey concretion was mainly composed of quartz, aragonite, and calcite while the reddish concretion was mainly composed of pyrite and quartz. Our study indicated that the formation process of the grey concretion probably included the crystallization and transformation of aragonite, while the corrosion of iron implements and crystallization of pyrite were highly involved in the formation of reddish concretion.     

A mixed microscopic method for differentiating seven species of “Bixie”‐related Chinese Materia Medica

Confusion in the species associated with the name of “Bixie” in Chinese Materia Medica began centuries ago. In recent decades, diverse medicinal plants from the genera Dioscorea and Smilax, and even minor species from the genus Heterosmilax, have been documented under the name Bixie or a very similar name as folk medicines in different areas of China. However, the traditional efficacies and chemical profiles of these herbs are not exactly the same and even vary wildly. Comprehensive authentication of multiple Bixie herbs has not yet been attempted. To differentiate and ensure the correct use of these Bixie‐related herbs, in this study, seven sorts of representative Bixie herbs (Dioscorea collettii, D. zingiberensis, D. nipponica, D. septemloba, Smilax china, S. glabra, and Heterosmilax japonica) were characterized based on the microscopic examination of their powders and cryotomed transverse sections. This is not only the first attempt to distinguish Bixie herbs by a comprehensive microscopic techniques, including common light microscopy, fluorescence microscopy, and polarized light microscopy, but also it is the first research to observe characteristics of transections of crude drugs under polarized lighting for the purpose of authentication. Polarized light has been found to provide a number of unique characteristics. The results indicate that starch granules, stone cells, vascular bundles, and other significant tissue features can be used to authenticate “Bixie” herbs. The method was proven to be quick, handy, specific, and simple. It should be widely applicable to other herbal materials. Microsc. Res. Tech. 77:57–70, 2014. © 2013 Wiley Periodicals, Inc.     

Distinguishing the Chinese materia medica Tiepishihu from similar Dendrobium species of the same genus using histological and microscopic method

The Chinese Materia Medica, Tiepishihu, used as a tonic for over one thousand years, is a well‐known precious medicine in China. According to the Chinese Pharmacopoeia, its source is the species Dendrobium officinale Kimura et Migo, which is distinguished from other species in Dendrobium genus. However, these species from the same genus are similar with Tiepishihu and caused confusion in the market. To find a quick and simple method to distinguish Tiepishihu from other similar species, histologic and microscopic methods were combined together to investigate the transverse section of stem of Tiepishihu and other similar species. Phloroglucinol test solution with hydrochloric acid was used to reveal the lignified tissue by staining the transverse section of Tiepishihu and similar species. Results revealed the unique identification characteristics to distinguish Tiepishihu from similar species, which were difficult to distinguish by other methods. The identification characteristics of Tiepishihu include the cells of vascular bundle sheath were stained red, parenchyma cells were not stained red. What's more, other species can be distinguished from each other with microscopic and histological characteristics. These characteristics proved stable and can be easily observed by normal light microscopic examination. This method is rapid, accurate, stable, and inexpensive.     

Microscopic analysis of “iron spot” on blue‐and‐white porcelain from Jingdezhen imperial kiln in early Ming dynasty (14th–15th century)

“Sumali,” as an imported cobalt ore from overseas, was a sort of precious and valuable pigment used for imperial kilns only, which produces characteristic “iron spot” to blue‐and‐white porcelain in early Ming Dynasty (A.D. 14th–15th century). Although there were some old studies on it, the morphology and formation of iron spot has not been fully investigated and understood. Therefore, five selected samples with typical spot from Jingdezhen imperial kiln in Ming Yongle periods (A.D. 1403–1424) were analyzed by various microscopic analysis including 3D digital microscope, SEM‐EDS and EPMA. According to SEM images, samples can be divided into three groups: un‐reflected “iron spot” without crystals, un‐reflected “iron spot” with crystals and reflected “iron spot” with crystals. Furthermore, 3D micro‐images revealed that “iron spots” separate out dendritic or snow‐shaped crystals of iron only on and parallel to the surface of glaze for which “iron spot” show strong metallic luster. Combining with microscopic observation and microanalysis on crystallization and non‐crystallization areas, it indicates that firing oxygen concentration is the ultimate causation of forming reflective iron spot which has a shallower distribution below the surface and limits crystals growing down. More details about characters of “iron spot” used “Sumali” were found and provided new clues to coloration, formation mechanism and porcelain producing technology of imperial kiln from 14th to 15th centuries of China.     

Study on morphological characteristics and microscopic structure of medicinal organs of Pulsatilla chinensis (Bunge) Regel

Modern clinical studies have found that Pulsatilla radix contains a variety of active ingredients; however, its medicinal parts and microstructure have been controversial. Based on morphological observation and microscopic identification of different structures, we chose Anhui, Henan, Jilin, and Shanxi Province to study Pulsatilla chinensis (Bunge) Regel populations. We specifically addressed different diameters and anatomical structures of different parts of underground organs. We also found that P. chinensis (Bunge) Regel had a “joint point” structure in its underground organ. Above the “joint point” is the rhizome, below the “joint point” is the root. The main medicinal organ is the rhizome. The protective tissues of the endothelial layer of P. chinensis (Bunge) Regel changed in the process of its development. The protective tissues were replaced by epidermis, cortex, endodermis, and phloem. With the secondary growth, the endothelial cells have been radial division. This study showed that morphology and microscopic identification was an important mean of medicinal material identifications, and it had the characteristics of a convenient, fast, and intuitive method to identify the composition and structural characteristics of P. chinensis (Bunge) Regel medicinal parts.     

A simple “hands‐off” apparatus to inflate concealed soft parts of the genitalia of small insect specimens

We describe a simple, effective and relatively cheap instrument for the inflation of soft parts of insect genitalia, particularly the vesica (endophallus) of small male lepidopteran specimens. It can also be applied in female genitalia and for the hyperextension of postabdominal segments of the telescoping type. A fine glass capillary is mounted on a self‐constructed micromanipulator system with capillary holder, moveably placed aside a microscope. The micromanipulator is constructed from the x‐ and y‐pinions of a discarded microscope table and the z‐pinion of a discarded microscope. Preparation is carried out as usual on a microscopic slide. The inflatable soft parts are pushed out mechanically and then fully inflated and subsequently hardened by pumping absolute alcohol with a medical syringe via a flexible PVC tube, the capillary holder and the glass capillary into the phallus. With affordable effort, our “hands‐off”apparatus might considerably ease preparation. Microsc. Res. Tech. 76:258–262, 2013. © 2013 Wiley Periodicals, Inc.     

Silver impregnation applied to striated muscle: The sarcoplasmic reticulum

The classical black reaction developed by Camillo Golgi is shown to impregnate the tubules and fenestrations of the sarcoplasmic reticulum (SR) in striated muscle. This is a double impregnation of chromate and silver, which usually fills extracellular spaces. The method is difficult insofar as long incubation times are required, and location of the successfully “stained” SR in plastic-embedded tissue blocks is unpredictable. The light microscope is absolutely necessary to find the good regions which can then be cut from the blocks in 1-μm-thick sections and examined in the electron microscope. Stereo pairs give the best results since these resolve overlap problems common to thick sections. A variety of artifacts are illustrated which can help avoid erroneous interpretations. The Golgi-“stained” SR shows this elusive network with unsurpassed contrast and should benefit the morphological studies of muscle-membrane enthusiasts.     

Authentication of herbal drug Tukhm‐e‐balango (Lallemantia royleana Benth.) using microscopic,pharmacognostic, and phytochemical characterization

The study is aimed to provide a comprehensive account on authentication of herbal drug named as Tukhm‐e‐balango (Lallemantia royleana Benth.) from the seeds of Ocimum basilicum by using microscopic, pharmacognostic, and phytochemical characterization. The crude medicinal plants and their parts are often adulterated or substituted in market due to improper identification by the consumers while among herbal plant sellers, taxonomic confusion is caused due to morphological similarities of the plant parts and lack of a standard identification system.In microscopy, both herbarium and fresh specimens were studied using qualitative and quantitative morphological characteristics of leaves, seeds, and pollen. For pharmacognosy, solubility, fluorescence, and physicochemical characterizers were analyzed whereas a total phenolic and flavonoids contents was determined in addition to DPPH radical scavenging activity. In current study, microscopic, pharmacognostic, and phytochemical characterization clearly differentiated L. royleana from O. basilicum. The major problem in herbal drug industry is caused due to confusion and controversy of certain synonyms used for more than one or two drugs. Sometimes, under the same common or local name, entirely different taxa are being sold in herbal markets. It is concluded that correct and proper identification of medicinal plants is very crucial to ensure the safety and efficacy of herbal medicines, as many medicinal plants are intentionally or unintentionally adulterated with similar species or varieties. In herbal market, the seeds of L. royleana are adulterated with seeds of O. basilicum due to their similar morphology.     

“Microscopic evidences of heavy metals distribution and anatomic alterations in breaching‐leaves of Cupressus lindleyi growing around mining wastes”

In this article a study of the distribution of heavy metals in Cupressus lindleyi breaching‐leaves was done in Taxco, Guerrero. At the same, heavy metals micro‐localization was conducted in the breaching‐leaves to understand the structural changes provoked by mining waste on plants. The most abundant contaminants in soils, tailings and different plant organs (roots, stems, and leaves) were Zn, Mn, and Pb. Nevertheless, As was more accumulated in the stem and breaching‐leaves. The translocation factor and the bio‐concentration factor were less than 1. The structural changes observed were the great accumulation of starch grains and phenolic compounds in the palisade parenchyma, changes in the hypodermis cell wall and necrotic zones in the palisade parenchyma. The distribution of heavy metals in breaching‐leaves tissues was homogeneous in most of the elements. These results showed that C. lindleyi is a species that can be employed in phytostabilization of contaminated zones with mining waste because it is a native plant that does not require a lot of conditions for its development. Microsc. Res. Tech. 77:714–726, 2014. © 2014 Wiley Periodicals, Inc.     

Structure and biomechanics of culms of Phragmites australis used for reeds of Japanese wind instrument “hichiriki”

Hichiriki is a traditional Japanese double‐reed wind instrument used in Japanese ancient imperial court music, gagaku, which has been performed since the 7th century. The best reeds for hichiriki have been made of culms or stems of Phragmites australis (P. australis) that are harvested from only a limited reed bed at Udono near Kyoto. The aim of this study is to elucidate why the stems from Udono are the best materials for hichiriki reeds. Plant anatomy was examined for choice stems of P. australis grown in different reed beds in Japan as well as morphology, and the local indentation hardness and Young's modulus of tissues on the cross‐sections of some representatives of hichiriki reeds were measured. It is concluded that the good stems for hichiriki reeds have an outer diameter of about 11 mm, a wall thickness of about 1 mm and comparatively homogeneous structure where harder materials, such as epidermis, hypodermis, sclerenchymatous cells, and vascular bundle sheaths with hard walls, are orderly deployed with softer materials such as parenchyma cells and vascular bundles. This structure has smaller differences of hardness and Young's modulus between the hard and soft materials in the reed, providing the best music performance. Microsc. Res. Tech. 78:260–267, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Using microscopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranunculaceae)

In present research, micromorphological characters of the genus Delphinium L. (sensu lato) Ranunculaceae seeds and fruits were studied using microscopic techniques. A total of 37 species were studied using light (LM) and scanning electron microscopy (SEM) to evaluate seeds and fruits micromorphological characteristics for the taxonomic identification and species relationship in the genus. For each taxon, 33 characters were observed and studied. All characters were analyzed using MVSP software and UPGMA method by using dissimilarity index (average taxonomic distance). Considerable differences were observed in trichomes shape of fruit and seed color features. The results revealed that all studied taxa are separated into three clades. The first clade consists of D. pallidiflorum and D. semibarbatum, which have black seed and two types of glandular and crispate trichomes in fruit. The second clade consists of D. speciosum, D. szowitsianum, D. dasystachyson, D. aquilegifolium, D. ursinum, C. trigonelloides, and C. camptocarpa, which had a brown color seeds, two types of glandular and villous trichomes on fruits also have been observed. The remaining species were classified as third clade having various colors including yellow, black, and brown color seeds, the trichome shapes included one of the following forms strigose, villous, trichomes less, glandular, hooked, and crispate trichomes in fruits. The aim of the present study is to use micromorphological characters of seeds and fruits for the taxonomic identification and species delimitation in the genus Delphinium.     

Ion mobility spectrometry‐mass spectrometry (IMS‐MS) of small molecules: Separating and assigning structures to ions

The phenomenon of ion mobility (IM), the movement/transport of charged particles under the influence of an electric field, was first observed in the early 20th Century and harnessed later in ion mobility spectrometry (IMS). There have been rapid advances in instrumental design, experimental methods, and theory together with contributions from computational chemistry and gas‐phase ion chemistry, which have diversified the range of potential applications of contemporary IMS techniques. Whilst IMS‐mass spectrometry (IMS‐MS) has recently been recognized for having significant research/applied industrial potential and encompasses multi‐/cross‐disciplinary areas of science, the applications and impact from decades of research are only now beginning to be utilized for “small molecule” species. This review focuses on the application of IMS‐MS to “small molecule” species typically used in drug discovery (100–500 Da) including an assessment of the limitations and possibilities of the technique. Potential future developments in instrumental design, experimental methods, and applications are addressed. The typical application of IMS‐MS in relation to small molecules has been to separate species in fairly uniform molecular classes such as mixture analysis, including metabolites. Separation of similar species has historically been challenging using IMS as the resolving power, R, has been low (3–100) and the differences in collision cross‐sections that could be measured have been relatively small, so instrument and method development has often focused on increasing resolving power. However, IMS‐MS has a range of other potential applications that are examined in this review where it displays unique advantages, including: determination of small molecule structure from drift time, “small molecule” separation in achiral and chiral mixtures, improvement in selectivity, identification of carbohydrate isomers, metabonomics, and for understanding the size and shape of small molecules. This review provides a broad but selective overview of current literature, concentrating on IMS‐MS, not solely IMS, and small molecule applications. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:43–71, 2013     

Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014. © 2013 Wiley Periodicals, Inc.