Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

Stereologic techniques in microscopy

The principal measuring methods employed in morphometry, generally known as stereology, allow information on volumes, surface areas, numbers of structures, and many other dimensions to be derived from simple counting operations. Until relatively recently these techniques have found only limited application in biology, although they have been used for many years in the inorganic sciences. With the development of reliable quantitative methods in physiology and biochemistry, however, stereologic techniques are becoming increasingly important and a number of interesting methods have been developed which are both rapid and simple. In this paper a number of practical techniques are presented which have proved useful in light and electron microscopy.     

Study of hyperpigmentation in human skin disorder using different electron microscopy techniques

There is a global trend of increase in the demand for three‐dimensional electron microscopy with high resolution. The ultrastructural change and related functional studies are necessary to investigate biological phenomena. In this study, currently available 3D reconstruction techniques of electron microscopes (serial block‐face scanning electron microscopy and focused ion beam—scanning electron microscopy) were used to investigate hyperpigmentary disorders in human skin. In the basal layer of the epidermis in the human skin, there are melanocytes that produce melanin and keratinocytes that act as a barrier against environmental damage. The 3D structure from serial images through scanning electron microscopy showed locations of melanosomes between melanocyte and keratinocyte in the hyperpigmentary disorder, in addition, the electron tomography showed pigment transfer through melanin instead melanosome. These results support the exocytosis‐endocytosis theory of pigment in human skin.     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ～300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ～300 nm to ～660 nm.     

Intraspecific variation in seed morphology of tribe vicieae (Papilionoidae) using scanning electron microscopy techniques

Seed micromorphology of 12 species of tribe Vicieae (Papilionoidae) representing five genera were examined using Scanning Electron Microscope (SEM). The different seed types were described, illustrated, compared, and their taxonomic importance is discussed. Seeds exhibit great diversity in ultrastructure and a variety of novel morphological features have been determined among and within genera of the tribe. Seeds were characterized by oval to round shape, ovate to oblong outline and striate to papillate ornamentation. Radiate and wavy cell pattern has been observed as a characteristic exomorphological feature. In Vicia sativa, testa cells superficially seem to form peltate hairs. As a result, seed micromorphology with few exceptions showed variation and its taxonomic value was significant in distinguishing taxa at species level. The disparity in shape and density of papillae or protuberances and alignment of testa cells, may possibly give further insight at intraspecific level within tribe. The findings also illustrated that the use of SEM in seed morphology can reveal hidden morphological affinities among species and showed potential in delimitation of Vicieae members generally at tribe, genus, and particularly at species level.     

Intraspecific variation in spermoderm pattern of tribe Acacieae (Mimosoideae) using scanning electron microscopy techniques

Current research carried out in Pakistan is the first report on spermoderm ornamentation of eight species of tribe Acacieae (Mimosoidae) by using scanning electron microscopic techniques representing two genera, Fedherbia and Acacia were examined. Different spermoderm ornamentation were observed, described and discussed for their taxonomic importance. Seeds surfaces of the studied tribe possess novel variations in macro and micro morphology. Great variations were observed in both qualitative and quantitative characters of seeds. Seeds shape was oblong, ovate to elliptical and spermoderm ornamentation was levigate, rugose, polygonal and discoid, colliculate, and papillose type. These variations in the spermoderm ornamentation can be used as an aid in identification and classification of the members of tribe Acacieae.     

Emission microscopy and related techniques: resolution in photoelectron microscopy, low energy electron microscopy and mirror electron microscopy.

A unified treatment of the resolution of three closely related techniques is presented: emission electron microscopy (particularly photoelectron microscopy, PEM), low energy electron microscopy (LEEM), and mirror electron microscopy (MEM). The resolution calculation is based on the intensity distribution in the image plane for an object of finite size rather than for a point source. The calculations take into account the spherical and chromatic aberrations of the accelerating field and of the objective lens. Intensity distributions for a range of energies in the electron beam are obtained by adding the single-energy distributions weighted according to the energy distribution function. The diffraction error is taken into account separately. A working resolution is calculated that includes the practical requirement for a finite exposure time, and hence a finite non-zero current in the image. The expressions for the aberration coefficients are the same in PEM and LEEM. The calculated aberrations in MEM are somewhat smaller than for PEM and LEEM. The resolution of PEM is calculated to be about 50 A, assuming conventional UV excitation sources, which provide current densities at the specimen of 5 x 10(-5) A/cm2 and emission energies ranging up to 0.5 eV. A resolution of about 70 A has been demonstrated experimentally. The emission current density at the specimen is higher in LEEM and MEM because an electron gun is used in place of a UV source. For a current density of 5 x 10(-4) A/cm2 and the same electron optical parameters as for PEM, the resolution is calculated to be 27 A for LEEM and 21 A for MEM.     

Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly- l -lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform^TM was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform^TM were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.     

On dark field techniques in transmission electron microscopy.

Different dark field techniques in transmission electron microscopy are investigated. In particular, a displaced aperture technique is developed, which produces high quality off-axis dark field images. The essential feature of the method is that the chromatic aberration of the objective and intermediate lenses is compensated for with the aid of a one-stage beam deflector in the object plane of the intermediate lens. In addition, the image distortion and the effect of the curvature of the image field are minimized by reducing the coefficient of sperical aberration of the intermediate lens. Diffracted beams, forming angles alpha up to 2.5 degrees with the optical axis, can be used for dark field imaging. Resolutions better than 30 A for alpha = 2 degrees, and 17 A for alpha = 1 degree, are obtained.     

Versatile controlling system for cryopreparation techniques in electron microscopy

A new setup for freeze-substitution and a versatile controlling system has been developed. Our goal was to build a simple system allowing precise control of the physical parameters of freeze-substitution experiments to learn more about their influences on the cellular ultrastructure and immunoreactivity of macromolecules. An improved apparatus for freeze-substitution, based on liquid nitrogen cooling, and a universal software for controlling the complex preparation protocols from cryofixation to final polymerization are described. This controlling system has the following advantages: it allows precise control and registration of temperature profiles, reconstruction of each individual step of previous experiments, and optimization of working conditions. The setup of the freeze-substitution apparatus is designed to run many different substitution media in parallel; freeze-substitution (cryostat), embedding (working platform), and polymerization are carried out at separate places; therefore, more experiments can be done simultaneously. The ergonomic working platform allows exchange of media at controlled temperature and easy handling; survey of the temperature in individual tubes is possible, and the system is protected from water condensation and uncontrolled warming by the deep freezer.     

Freeze-substitution techniques for preparing nematodes for scanning electron microscopy

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO₄/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.     

Design and evaluation of a mechanical nanomanufacturing system for nanomilling

This paper presents design and evaluation of a mechanical nanomanufacturing system for performing the nanomilling process. The nanomilling process uses a nanotool (an atomic force microscope probe tip) that is rotated at high speeds to fabricate three-dimensional (3D) nano-scale features on a sample surface. After explaining the kinematics of the two nanomilling process configurations, the nanomilling system, including the 3D piezoelectric actuator that rotates the nanotool, the nanopositioning stage that provides the feeding and depth motions, and the software program that controls the nanomilling motions are described. A measurement system is then constructed to measure the dynamic nanomilling motions. A compensation algorithm is developed to enable obtaining desired nanotool motions in the presence of frequency and amplitude-dependent nonlinearities of the 3D piezoelectric actuator. The nanomilling system is then evaluated directly by measuring the nanotool motions, and indirectly by assessing the accuracy of the fabricated nanoscale features. It was shown that the nanomilling system facilitates fabrication of complex nano-scale features with high accuracy through the high-stiffness nanotool assembly and high-frequency (compensated) nanotool motions.     

Evaluation of scanning electron microscopy images of a model dressing using image feature extraction techniques and principal component analysis

Twelve dressing systems made by varying protein type, oil level, CaCl₂, NaCl, and sucrose, were examined using scanning electron microscopy. Images from the 12 systems were quantitatively analysed using methods of feature extraction. These methods were based on vectorisations of the images followed by principal component analysis on the extracted vectors. These techniques were used to examine the reproducibility of the acquired images as well as to relate the images to rheologic and sensory texture parameters. Two feature extraction methods were used: the angle measure technique (AMT) and the absolute difference method (ABDF). The ABDF method used fewer principal components to extract information from images relevant to the complex modulus/sensory viscosity of the system, but the information seemed equally well preserved by the two-feature extraction methods. The AMT was more efficient in classifying the images with respect to protein type. A fair correlation between images and complex modulus was obtained (R=0.73). It is suggested that a better correlation might be obtained by adding more systems, increasing the number of areas imaged for each system as well as avoiding systems of low viscosity.     

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.     

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.     

A study on using scanning acoustic microscopy and neural network techniques to evaluate the quality of resistance spot welding

This paper shows that the quality of resistance spot welds can be evaluated using scanning acoustic microscopy (SAM). Two-layered coated spot-welded samples are investigated utilising a wide-field short-pulse scanning acoustic microscope with operation frequencies of 25, 50 and 100 MHz. Geometrical parameters, e.g. nugget area, maximum axis of nugget, and minimum axis of nugget, are acquired from C-scan images of weld nuggets using mathematical morphology techniques. These parameters serve as inputs for an artificial neural network (ANN) model to evaluate the quality of spot welds. The output of the model during the training process comprises the results of nugget peeling tests and expert opinions. The ANN can provide suggestions on weld quality with a higher than 95% correctness. A JAVA computer program is developed for image processing, ANN training, and ANN testing. With this model, the computer program can render the quality of spot welds that are close to those achieved using off-line destructive method.     

Time-lapse FRET microscopy using fluorescence anisotropy

We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein–protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.     

Variations in fungal spore dimensions in relation to preparatory techniques for light microscopy and scanning electron microscopy

The volumes of urediospores of Uromyces viciae-fabae and ascospores of Sordaria humana in the critical point-dried, freeze-dried, osmium vapour-fixed/freeze-dried, frozen-hydrated and ‘wet’ states were measured. Dried spores shrank and were significantly smaller than frozenhydrated and ‘wet’ spores. Urediospores exhibited a greater range of dimensional changes in relation to the preparatory treatment than did ascospores. Ascospores, dried in different ways, were not significantly different from each other but were significantly smaller than frozenhydrated ones. Both urediospores and ascospores dehydrate naturally and collapse. On wetting they imbibe water rapidly and swell. Low temperature scanning electron microscopy may be suitable for investigating fungal spores in their ‘natural states' on the leaf surface.