Synchrotron nanoscopy imaging study of scalp hair in breast cancer patients and healthy individuals: Difference in medulla loss and cortical membrane enhancements

Nanoscopic synchrotron X‐ray imaging was performed on scalp hair samples of patients with breast cancer and healthy individuals to investigate any structural differences as diagnostic tool. Hair strands were divided into 2‐3 segments along the strands from root to tip, followed by imaging either in projection or in CT scanning with a monochromatic 6.78‐keV X‐ray using zone‐plate optics with a resolving power of 60 nm. All the examined cancer hairs exhibited medulla loss with cancer stage‐dependent pattern; complete loss, discontinuous or trace along the strands. In contrast, medullas were well retained without complete loss in the healthy hair. In the CT‐scanned axial images, the cortical spindle compartments had no contrast in the healthy hair, but appeared hypointense in contrast to the surrounding hyperintense cortical membrane complex in the cancer hair. In conclusion, observation of medulla loss and cortical membrane enhancements in the hair strands of breast cancer patients demonstrated structural variations in the cancer hair, providing a new platform for further synchrotron X‐ray imaging study of screening breast cancer patients. Microsc. Res. Tech. 79:23–30, 2016. © 2015 Wiley Periodicals, Inc.     

Fluorescence microscopy study on the cytoskeletal displacements during sperm differentiation in the bush‐cricket Tylopsis liliifolia (Fabricius) (Orthoptera: Tettigoniidae)

A study by fluorescence microscopy has been carried out on male gametes from testicular follicles, seminal vesicles, spermatophores, and seminal receptacles of the bush‐cricket Tylopsis liliifolia, focusing the attention on localization and movements of F‐actin and α‐tubulin during sperm differentiation, since data in this respect are lacking in the Orthoptera. F‐actin and α‐tubulin positivity was detected in the testicular follicles, in particular at the bridges connecting spermatids of a same clone and around their nucleus, during the first differentiation stages. During the following differentiation stages in the testes, F‐actin was found at one of the spermatid poles and then, during nucleus elongation, at the whole acrosomal region. A peculiar F‐actin‐positivity was found at the flagellum, more markedly immediately posterior to the nucleus, at the basal body region of the gametes from the testicular follicles and from the other examined districts. Other interesting data from our investigations concerns the α‐tubulin displacements during the differentiation stages of the spermatid and a constant absence of α‐tubulin‐positivity where the centrioles are located. No positivity was also found for both α‐tubulin and nuclear markers at the anterior region of the gamete, where the acrosomal wings are localized. Our results, compared with what is so far known in literature for the insects, lead us to assert that microfilaments and microtubules undergo gradual displacements, markedly in the testicular follicles, during the morphogenesis of the male gamete of T. liliifolia aimed to its organization and motility and probably also to its interaction with the female gamete. Microsc. Res. Tech. 79:81–88, 2016. © 2015 Wiley Periodicals, Inc.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

Organization of microtubules in cochlear hair cells

The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.     

Returning to the same area of hair surfaces before and after treatment: a longitudinal AFM technique

We report the use of longitudinal (aspect ratio > 1 : 1) scanning atomic force microscopy as an aid in returning to the same area of hair fibres after bleaching, treatment with a commercial shampoo or the application of a ‘leave‐on’ conditioner product. The bleaching treatment used in this study was not found to affect the cuticular architecture and lateral force microscopy (LFM) also showed little difference after treatment, reflecting the homogeneity of the newly revealed surfaces. After treatment with a commercial shampoo, the hair sample again showed very little difference in topography or lateral force characteristics. Hair treated with the leave‐on conditioner product also showed no major topographical changes. LFM traces, however, showed regions between the ghost edge, marking the original position of the scale edge before cuticular erosion, and the existing scale edge, to have higher frictional properties than distal regions of the cuticle. A thin film of the leave‐on product thus seems to form in this region and extends from the foot of the scale edge.     

Atomic force microscopy of human hair

The atomic force microscope (AFM) was used to investigate the surface architecture of the entire lengths of cleaned human head hairs. Many features previously seen with the scanning electron microscope (SEM) were identified. However, the AFM has provided much greater detail and, in particular, the hair's cuticular surfaces appear not to be as smooth as had been previously supposed. A consistent feature was of step discontinuities or "ghosts" on the scale surfaces. These delineated the original location of each overlying scale before its edge had been chipped away. There was a change in the longitudinal angular presentation of the surfaces about each ghost. This means the distal ends of each cuticle cell have been synthesised in the follicle to be thicker than where that same cuticle cell is bounded on both sides by other cuticle cells. The undamaged outer cuticular surfaces at the root end of each hair were covered everywhere by longitudinal ridges (striations). Where the hair surface was worn, the striations terminated at a scale edge ghost. The ridges were approximately 9 nm high and were in parallel array with a lateral repeat spacing of about 350 nm. The striations are evidently formed on the outer surface of each cuticle cell following earlier contact in the hair follicle with the inner root sheath. The study of stained transverse sections of hairs in the transmission electron microscope (TEM) is suggested as a means for throwing some light on the underlying structure and chemistry of the striations. Finally, our AFM studies have revealed that the surface of the freshly emergent hair gradually changes over a distance of about 20 mm and that the surface of the hair for most of its length is quite different from that near the root. This is likely to be of import to those engaged in the hair toiletries industry.     

A scanning electron microscopic study on the cephalic cuticular sensillae of different larval stages of Antheraea assamensis,an indigenous silkworm species of Northeast India

Scanning electron microscopy revealed the presence of a number of cuticular sensillae on the head of different larval stages of the “Muga” silk moth, Antheraea assamensis. These include four types of sensilla trichodea, bead‐like folded sensilla, two types of sensilla basiconica, and sensory pegs. The study seems to be the first attempt in describing the morphology and distribution pattern of the cephalic cuticular sensillae of different larval stages of the silk moth Antheraea assamensis, an indigenous species of Northeast India. The possible significance of the sensillae in searching behavior of the larval stages during feeding is discussed with the help of available literature. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

Light and electron microscopy study of the spermatheca of Eupholidoptera chabrieri bimucronata (Ramme, 1927) and Uromenus brevicollis trinacriae La Greca 1964 (Orthoptera: Tettigoniidae)

A study by both optical and electron microscopy has been carried out on the spermatheca of Eupholidoptera chabrieri bimucronata and Uromenus brevicollis trinacriae (Orthoptera: Tettigoniidae). In both the examined species, the spermatheca consists of a sac/kidney‐shaped seminal receptacle and a more or less tortuous spermathecal duct that opens into the common oviduct. The wall of both the organs consists of a pseudostratified epithelium surmounted by a cuticular intima; the latter is made up of a thicker endocuticle and an epicuticle. The epithelium shows two different cell types, irregularly arranged and with well differentiated functions: cuticle‐forming and gland cells. In both the species, the cuticle‐forming cells perform other functions, in addition to producing the cuticular intima. The gland cells never come in contact with the cuticular intima, have inside the reservoir a secretion whose appearance can diversify also in contiguous zones of the seminal receptacle. Based on our findings in both the species, the functions of the seminal receptacle would differ from those of the spermathecal duct. In the latter, some areas of the wall of the connecting tract show an activity of lysis, by contiguous epithelial cells, that could play a role in control and selection of spermatozoa. As for the feather‐shaped spermatodesms, similar in both the species, freeze‐fracture observations have shown that the acrosome of each spermatozoon regularly covers three‐quarters of the extension of the acrosome of the following spermatozoon. Finally, the significance of our findings, compared with what is known in literature, is discussed. Microsc. Res. Tech. 78:577–586, 2015. © 2015 Wiley Periodicals, Inc.     

Physiological significance of gravity receptors on larval cephalic cuticle in the silk moth,Antheraea proylei Jolly

The lateral aspects of larval cephalic cuticle of oak tasar moth, Antheraea proylei, a hybrid between Antheraea pernyi and Antheraea roylei exhibited the presence of gravity receptors in the form of dorsal campaniform sensilla. The distribution pattern and number of dome shaped dorsal campaniform sensilla were found to vary in different larval stages. In the first and second larval stages, 4‐5 sensilla were localized near the apex of the lateral aspects of the cephalic cuticle on either side of the head. From the third larval stage onwards, on the other hand, both the right and left lateral aspects of the cephalic cuticle were covered with innumerable dome shaped dorsal campaniform sensilla. The sensilla were found to be arranged in groups of 3 to 5 on the cephalic cuticle with dome‐free cuticular portion of about 50–100 µm in length between two adjacent groups of the sensilla. The individual dome shaped dorsal campaniform sensillum was either smooth surfaced or was covered with smaller domes through out the surface. The significance of differences in the number and distribution pattern of the dorsal campaniform sensilla among different larval stages in relation to gravity reception and preferred feeding posture are discussed in the light of available literature.     

In vivo second‐harmonic generation and ex vivo coherent anti‐stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb‐fiber laser‐based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 2015. © 2015 Wiley Periodicals, Inc.     

Morphology of the third instar larva of Colobopterus quadratus (Coleoptera: Scarabaeidae: Aphodiinae) using scanning electron microscopy

Aphodiinae are a large cosmopolitan subfamily of Scarab beetles (Scarabaeoidea: Scarabaeidae), playing an important role during dung decomposition. Many aspects, especially immature stages, of these dung beetles have not yet been recorded due to their comparative small body sizes and lacking of striking rolling behavior. In this study, the third instar larva of Colobopterus quadratus is morphologically described using scanning electron microscopy. The larva is atypical for the combination of following morphological features: (a) the larval antennae are five‐segmented; (b) the clypeus bears 4 minute protuberances on dorsal surface; (c) the epitorma on epipharynx is ventrally protuberant; (d) the scissorial area of left mandible is broad and blade‐like; (e) the raster is furnished with six glabrous protuberances and two rows of pali arranged in V‐shape. Some significant ultra‐morphological features were discovered in larval Aphodiinae for the first time. The presence of blade‐like incisor on the left mandible implies the potential root cutting ability.     

Nanomechanical characterization of human hair using nanoindentation and SEM

Human hair is a nanocomposite biological fiber with well-characterized microstructures. Nanomechanical characterization of human hair can help to evaluate the effect of cosmetic products on hair surface, can provide a better understanding of the physicochemical properties of a wide variety of composite biological systems, and can provide the dermatologists with some useful markers for the diagnosis of hair disorders. The paper presents systematic studies of nanomechanical properties of human hair including hardness, elastic modulus and creep, using the nanoindentation technique. The samples include Caucasian, Asian and African hair at virgin, chemo-mechanically damaged and treated conditions. Hair morphology was studied using scanning electron microscopy (SEM). Indentation experiments were performed on both the surface and cross-section of the hair, and the indents were studied using SEM. The nanomechanical properties of hair as a function of hair composition, microstructure, ethnicity, damage and treatment are discussed.     

Investigation of dyed human hair fibres using apertureless near-field scanning optical microscopy

We present the first studies of dyed human hair fibres performed with an apertureless scanning near‐field optical microscope. Samples consisted of 5‐µm‐thick cross‐sections, the hair fibres being bleached and then dyed before being cut. Hair dyed with two molecular probes diffusing deep inside the fibre or mainly spreading at its periphery were investigated at a wavelength of 655 nm. An optical resolution of about 50 nm was achieved, well below the diffraction limit; the images exhibited different optical contrasts in the cuticle region, depending on the nature of the dye. Our results suggest that the dye that remains confined at the hair periphery is mainly located at its surface and in the endocuticle.     

Hyperspectral interferometry: Sizing microscale surface features in the pine bark beetle

A new method of interferometry employing a Fabry–Perot etalon model was used to locate and size microscale features on the surface of the pine bark beetle. Oscillations in the reflected light spectrum, caused by self‐interference of light reflecting from surfaces of foreleg setae and spores on the elytrum, were recorded using white light hyperspectral microscopy. By making the assumption that pairs of reflecting surfaces produce an etalon effect, the distance between surfaces could be determined from the oscillation frequency. Low frequencies of less than 0.08 nm^?1 were observed in the spectrum below 700 nm while higher frequencies generally occupied wavelengths from 600 to 850 nm. In many cases, two frequencies appeared separately or in combination across the spectrum. The etalon model gave a mean spore size of 3.04 ± 1.27 μm and a seta diameter of 5.44 ± 2.88 μm. The tapering near the setae tip was detected as a lowering of frequency. Spatial fringes were observed together with spectral oscillations from surfaces on the exoskeleton at higher magnification. These signals were consistent with embedded multi‐layer reflecting surfaces. Possible applications for hyperspectral interferometry include medical imaging, detection of spore loads in insects and other fungal carriers, wafer surface and subsurface inspection, nanoscale materials, biological surface analysis, and spectroscopy calibration. This is, to our knowledge, the first report of oscillations directly observed by microscopy in the reflected light spectra from Coleoptera, and the first demonstration of broadband hyperspectral interferometry using microscopy that does not employ an internal interferometer. Microsc. Res. Tech. 78:873–885, 2015. © 2015 Wiley Periodicals, Inc.     

The occurrence of autophagic cell death in the tegument of rabbits pre‐infested with Rhipicephalus sanguineus and exposed to selamectin (active principle of acaricide pfizer revolution®)

Ticks of Rhipicephalus sanguineus species have great medical and veterinary importance for being a vector of various diseases. In an attempt to minimize their action on the host, people have resorted to chemical control by using various acaricides, such as selamectin. Although previous studies have demonstrated its toxic action in domestic animals, no studies focused on the detection of cell death when exposed to selamectin. For this reason, the technique for detecting autophagic cell death was used in order to demonstrate the responses of rabbits' skin tissues pre‐infested with R. sanguineus and exposed to different concentrations of selamectin. The obtained results when exposed to 100 and 80% concentrations of selamectin showed a strong mark of acid phosphatase on the cells of the connective tissue of the dermis and hair follicles, whereas the ones exposed to the 50% concentration had a weak mark on the cells of the connective tissue of the dermis and moderate staining in hair follicles. It became clear that, when used at high concentrations (100 and 80%), selamectin is capable to induce a large scale occurrence of the autophagic cell death process. On the other hand, the concentration of 50% causes minor morphophysiological changes in the skin of rabbit hosts when evaluated the cell death process. Therefore, the data confirms that selamectin is a powerful dose‐dependent toxic agent causes increased activity of the enzyme acid phosphatase. Microsc. Res. Tech. 76:1171–1176, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of atopic dermatitis on the morphology and water content of scalp hair

The effects of atopic dermatitis (AD) on scalp hair properties, such as morphology and water content, were investigated using atomic force microscopy (AFM) and thermogravimetric analyzer. Hairs from lesional and nonlesional scalp regions of eight patients with AD were investigated. The severity of the disease, which was evaluated using the SCORing Atopic Dermatitis index, was 48.75 (range, 40-80). Hairs from 15 normal adults were also examined as controls. The surface images were taken in an area of 20 × 20 μm(2) with 512 × 512 pixels and a scan speed of 0.8 line/s. AD affected the cuticle structures and scales of scalp hair. The edges of cuticles were torn and collapsed, and the scales were very thick. The water contents of both types of AD hair were less than the control: 12% ± 0.7%, 11.7% ± 0.4%, and 13% ± 0.8% for lesional AD hair, nonlesional AD hair, and control hair, respectively. The scalp hair of patients with AD can be characterized by thick and globular scale patterns. The hair of patients with AD has less water content than normal hair showing a good agreement with the property of skin having AD.     

Computerized morphometry analysis of epithelial fimbriae nuclear symmetry in BRCA carriers may identify patients at risk for developing ovarian cancer

Serous ovarian tumors may originate in epithelial cells of the fallopian tubes. Computerized morphometry was able to find significant alterations in the fallopian tube epithelium of healthy BRCA carriers. The purpose of this study was to identify a subgroup of BRCA carriers that may be at risk of developing serous ovarian cancer by evaluation of the epithelial nuclear symmetry in the fallopian tubes. Four groups of patients were analyzed; healthy patients, ovarian cancer patients, BRCA carriers, and BRCA noncarriers. All fallopian tubes appeared normal by H&E examination. The ImageProPlus software was used to assess the nuclear symmetry of 65 fimbriae epithelium cells and an artificial neural network algorithm aided in detecting a subpopulation among fimbriae of healthy BRCA carriers at risk for ovarian cancer. Significant differences were found between healthy patients and ovarian cancer patients, and between BRCA carriers and noncarriers. The algorithm was able to accurately predict BRCA carriers with associated ovarian cancer based on fallopian tube nuclear symmetry characteristics. These results reinforce the hypothesis that fimbriae epithelial cells of BRCA carriers may undergo early-stage changes that could predict the risk of progression toward malignancy.