In vivo observation of the corneal epithelium

Specular microscopy is an important addition to the ophthalmologist's diagnostic armamentarium. Using this technique, it has been found that normal corneal epithelial cells are polygonal, with no abnormal specular reflex. There are no spindle-shaped, large or small cells, as may be observed in a variety of pathologic conditions. Spindle-shaped cells are characteristic of the wound-healing process with cell migration. Large cells were observed not only in wound healing, but also in aphakic diabetic patients, extended-wear soft contact lens wearers, and individuals with keratoconus. These changes may result from depressed mitosis or inhibited sloughing of superficial cells. Morphometric analysis adds a quantitative dimension to epithelial cell analysis.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.     

In vivo assessment of emphysema in mice by high resolution X-ray microtomography

High resolution X-ray microtomography (micro-CT) was used for the detection of emphysema in live mice. Emphysema was induced in C57BL/6 J mice by intratracheal instillation of different amounts of porcine pancreatic elastase. This emphysema could be clearly detected by micro-CT seven weeks post-treatment: analysis of the whole data set of virtual cross-sections showed the presence of a dose-dependent level of emphysema.     

In vivo response to HA-polyhydroxybutyrate/polyhydroxyvalerate composite

This study examined the morphological and compositional structure of bone–implant interfaces after in vivo implantation into the tibias of rabbits. The implants were composed of biodegradable polyhydroxybutyrate/polyhydroxyvalerate copolymer reinforced with synthetic hydroxyapatite (HA) particles. Optical and scanning electron microscopy techniques were used, including energy‐dispersive X‐ray analysis. The interface was found to be morphologically, biologically and chemically active throughout the period of study. There was a strong tendency to rebuild the bone structure at the interface after implantation, independent of the composition of the implant, but direct bone bonding with the implant depended on the bioactive nature of the interface, as represented by the HA particles. At all implantation times, lamellar bone formed at the interface and replaced degrading polymer matrix, while engulfing HA filler particles. In regions about 50–100 from the interface, the bone region displayed an osteon organization. Osteoblasts and osteocytes were identified throughout the interface region. The thickness of the newly formed bone significantly increased over the period of the experiment from about 130 µm at 1 month to about 770 µm at 6 months. Materials that behave in this manner may be useful in some bone replacement therapies.     

用激光扫描共聚焦显微镜原位检测细胞凋亡

用激光扫描共聚焦显微镜在原位检测细胞凋亡具有多方面的优势。本文介绍用该仪器原位检测Annexin—Ⅴ试验样品、TUNEL试验样品及进行细胞凋亡形态学观察的过程和方法。     

Image processing techniques in a preliminary morphometric characterization of corneal epithelium by scanning electron microscopy

Several processing techniques of digital images allowed us to quantify the percentage of cell surface covered by microprojections (microvilli or microplicae) (SCM), the adhesion between epithelial cells by the parameter intercellular junctions (IJ), the size (cell area), shape (cell shape) and shade (cell shade) of cells on the corneal epithelium of nine rabbits. The data were analyzed and the epithelial cells were classified into three groups by cluster analysis. Assuming the representativeness of the sample, our findings suggest that for a normal corneal epithelium, 80% of the cells could show SCM >41%, and IJ >0.98 (being one a cell to cell junction without disruptions). Standard deviations of cell shade lower than 21 gray levels could indicate a tendency to lose the cell shade mosaic. Normal corneas could show a majority of cells (54–69%) included in group 2 with smaller mean size (80% of cells with cell area <242 μm²), higher SCM (80% of cells with SCM >44.83%), polygonal mean shape and brighter shade. Group 1 (15–30% of cells) could show a larger mean size (80% of cells with cell area >494 μm²), lower SCM (although 80% of cells with SCM >32.61%), circular mean shape and darker electron reflex. Group 3 could display a medium mean size, higher SCM (similar to group 2), circular mean shape (similar to group 1), and brighter shade. These analyses could possibly be used to further assess the corneal response to ocular drugs, contact lens, and surgical procedures or to discriminate between pathology stages. Microsc. Res. Tech. 73:1059–1066, 2010. © 2010 Wiley‐Liss, Inc.     

In-situ observation and relocation method of nanomaterial samples based on microscope systems

Taking poly(lactic acid) microbubbles and purple membranes as examples, a general in situ observation and relocation method of nanomaterial samples based on microscope systems was reported. First, a four-grade coordinate with different precisions was marked around a substrate by UV lithography. Second, using optical microscope and scanning probe microscope, special positions of poly(lactic acid) microbubbles and purple membranes were observed, respectively. Third, the four-grade coordinate value corresponded to the special sample position, and the distance between the special position and coordinate edge were recorded, respectively. Finally, the special position can be easily found again, or the sample in the special position can be manipulated and secondary processed based on the recorded coordinate value and distance, after the sample was removed and then was reset on the sample stage of microscope. The in situ observation and relocation method can be applied in different microscope systems and different sample substrates, and will have potential applications in the manipulation and the secondary process of micro- and nano-devices.     

Three-dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.     

A case of Leukonychia with scanning electron microscope observation

Leukonychia is a medical term for white discoloration appearing on nails. The pathophysiologic mechanisms that cause white discoloration are not entirely clear. We processed a case of leukonychia with scanning electron microscope observation and found many crispy, obviously dissociated "layers" in the lower part of the white nail plate. The dissociated "layers" were composed of thick, loose, coarse keratin bundles intertwined with each other. We believe the dissociated "layers" are related to the clinically noted white discoloration appearing on the nails.     

A prototype two-detector confocal microscope for in vivo corneal imaging

Confocal microscopy through-focusing (CMTF) of the cornea produces a three-dimensional (3-D) display of corneal structure and intensity profiles that allow objective measurements of corneal sublayer thickness and relative assessment backscattering of light. In this study, a prototype confocal instrument was evaluated in which a photon counting photomultiplier tube (PMT) detector was added to provide faster and more quantitative measurements, while still maintaining the imaging capability of the microscope. To acquire images and measure backscattered light simultaneously, an uncoated pellicle beam splitter was incorporated into the light path of the confocal microscope. This beam splitter reflects 8% of the confocal signal to the PMT. The CMTF scans were performed on four rabbits using the prototype instrument. Corneal images and 3-D reconstructions acquired with and without the beam splitter in the light path appeared identical. Both the camera and PMT CMTF curves had easily identifiable peaks corresponding to the epithelium, basal lamina, and endothelium. No significant differences were found between PMT and camera CMTF measurements of epithelial, stromal, or corneal thickness (n = 12 scans). Furthermore, a high correlation was found between camera and PMT measurements (linear regression analysis, y = 0.999 x -0.4, r = 0.99, p < 0.001). The data suggest that by adding a pellicle beam splitter, CMTF intensity data can be acquired using a PMT. The PMT has a faster sampling rate and greater dynamic range than the camera and provides a count of the photons detected. Thus, the instrument has the potential for improving corneal pachymetry and back-scattering measurements while still providing high-resolution corneal images.     

In vivo spatio-temporal visualization of the human skin by real-time confocal microscopy

A modified tandem scanning confocal microscope is used to obtain in vivo images of the human skin in real time. Three-dimensional and temporal visualizations are demonstrated with volume reconstruction and blood flow images. Two image processing methods based on Fourier transform and logarithmic processing are presented. Their applications in noise removal of the scanning disk lines and of the heterogeneity of light are illustrated.     

In vivo coherent anti-Stokes Raman scattering imaging of sciatic nerve tissue

We report in vivo nonlinear optical imaging of mouse sciatic nerve tissue by epidetected coherent anti‐Stokes Raman scattering and second harmonic generation microscopy. Following a minimally invasive surgery to open the skin, coherent anti‐Stokes Raman scattering imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibres were demonstrated with high signal‐to‐background ratio, three‐dimensional spatial resolution, and no need for labelling. The underlying contrast mechanisms of in vivo coherent anti‐Stokes Raman scattering were explored by three‐dimensional imaging of fat cells that surround the nerve. The epidetected coherent anti‐Stokes Raman scattering signals from the nerve tissues were found to arise from interfaces as well as back reflection of forward coherent anti‐Stokes Raman scattering.     

In vivo confocal microscopy of human skin: A new design for cosmetology and dermatology

In-depth exploration of cellular structures in living human skin in situ is possible with the tandem scanning microscope (TSM). However, the rigid design of the microscope limited observations to the arms, hands, and fingers. A mobile version allowing the investigation of any parts of the body has been designed. The head containing the Nipkow disk and the optical path were the only part saved from the original TSM. This prototype can be used to observe, in real time, the different skin structures down to a depth of 200 μm and to measure the thickness of the different layers with micron precision level. The hydration of the stratum corneum (SC) could be assessed. For example, lengthy immersion of the hand in water led to an increase in SC thickness without affecting that of the living epidermis. Occlusive patch tests also showed that water and, even more so, propylene glycol, led to transient swelling of the SC. In dermatology, the example of psoriasis illustrated the value of the TSM for describing, measuring, and assessing pathologic skin changes. The availability of this noninvasive method for observing changes with time in a given skin site should prove useful for monitoring treatment efficacy. This tool opens up new insight for the investigation of cutaneous pathophysiology.     

Development of high-resolution field emission scanning electron microscopy with multifunctions for chargeless observation of nonconducting surface

We have developed a fully digital field emission scanning electron microscope (FE-SEM) with multifunctions to compensate the charging up of nonconducting surfaces. High-voltage observation, minimum electron dose, variable scanning speed, averaging, integration, tuning of surface potential, and cyclotron movements of secondary electrons have been achieved. This FE-SEM was successfully applied to observe resist, diatomaceous earth, aluminum oxide, and zeolite surfaces. The accelerating voltage is changeable in a range from 0.5 to 30 kV, and the probe current on the sample can be varied from 2×10^-9 to l×10^-13A to supply optimum electron dose. By using a snorkel- type, strongly excited objective lens (OL) immersing the samples in the magnetic field, the secondary electrons are extracted from the sample. For guiding electrons into the built-in lens-type secondary electron detector (SED), newly developed accelerating and retarding electrodes are installed in the OL to tune the surface potential. Furthermore, this FE-SEM can select 10 scan speeds, and the averaging and integration of secondary electron image signals are possible under every selected scan speed.     

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s^?1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.     

Optimisation of the environmental scanning electron microscope for observation of drying of matt water-based lacquers

Environmental scanning electron microscopy (ESEM) modifies conventional SEM through the use of a partial gas pressure in the microscope specimen chamber. Like conventional SEM, it has the resolution to image structure on the submicron lengthscale, but can also tolerate hydrated specimens if water vapour is used in the specimen chamber. This ability to image aqueous specimens leaves ESEM uniquely placed to study in situ drying in polymer latexes. However, there are two key practical difficulties associated with in situ drying. First, the size of the latex particles: larger latex particles are typically around 500 nm in diameter. Although ESEM can resolve structure on this lengthscale without difficulty, the magnification required results in radiation damage of the specimen due to the electron beam. This means that a given region can be imaged only once during film formation, so the evolution of particular features cannot be followed. Second, the change from ambient temperature and pressure to the ESEM conditions of 7 degrees C and 7.5 torr (100 Pa) can subject the specimen to a very high evaporation rate, which can disrupt film formation. The inclusion of a drop of water in the specimen chamber is shown largely to alleviate this, enabling successful imaging of film formation in the lacquer. Instead of the polymer latex itself, this work concentrates on a matting lacquer with silica inclusions. The silica matting agent particles are 1-10 microm in size, allowing for a lower magnification to be used, massively reducing specimen damage. Furthermore, the contrast during drying is much enhanced in the presence of silica. The images reveal the silica as bright regions against a darker background of polymer and water. Film formation shows the transition from a uniform, featureless aqueous solution to a polymer film with silica particles present on the surface. The appearance of individual silica particles can be followed. The particles are generally revealed quite early, after a few minutes of drying time. As film formation progresses, these same particles appear larger and more distinct. Few new particles are revealed at longer film formation times.     

斑马鱼卵表面形貌的环境扫描电镜观察

用环境扫描电子显微镜对斑马鱼卵的表面形貌直接进行观察。通过对环境条件的比较,认为在0℃左右,气压为4.39×102Pa时对鱼卵进行观察不发生脱水、放电等影响观察的现象。通过观察发现在鱼卵表面存在许多大小不一的突起,在突起附近还有一些丝状物。当鱼卵在有机农药中接受污染12h后,表面的突起变小,且丝状物消失。     

Use of the environmental scanning electron microscope for the observation of the swelling behaviour of cellulosic fibres

We have developed a method for observing transverse swelling of cellulosic fibres in the environmental scanning electron microscope (ESEM). The presence of liquid water in the ESEM specimen chamber allows the observation of in situ hydration without the need for coating, freezing, or drying of the sample. For reproducibility of the hydration and dehydration process, specialised mounting techniques are required and control of the conditions for condensation and evaporation of liquid water is necessary. The sensitivity of these cellulosic materials to the electron beam was investigated, showing that some damage mechanisms are enhanced by the continual presence of water vapour in the chamber. A discussion is presented of the effect of various experimental parameters on the extent and time of onset of the damage, and we outline steps to maximise the amount of useful experimental time for these fibres.     

Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating voltage of E = 1 keV and an ion-beam dose of It = 360 ～ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ～ 2 keV and It = 500 μA. min.