Wide field scanning slit in vivo confocal microscopy of flattening-induced corneal bands and ridges

We used a wide field scanning slit confocal microscope to examine the response of the in vivo human cornea to flattening. Flattening-induced effects consisted of (1) anterior corneal mosaic, which appeared as a meshwork of intersecting stromal and Bowman's layer bands with overlying epithelial ridges; (2) deep and middle stromal bands, which were narrower than and unrelated in position to the anterior corneal mosaic; and (3) posterior surface ridges. The posterior surface ridges projected posteriorly into the anterior chamber consisted of endothelium, Descemet's membrane, and posterior stroma, and were unrelated in position to posterior stromal bands. Confocal microscopy is a promising modality in the examination of the cornea and its response to mechanical stress.     

Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.     

Unique features of the basal cells of human prostate epithelium

The prostate gland is globally composed of epithelium and stroma. The epithelium plays an important role in the development of both benign and malignant disorders while the stroma is involved in benign prostatic hyperplasia. While the prostatic epithelium of the majority of laboratory animals is well recognized as a pseudostratified columnar, the classification of the human prostatic epithelium is controversial. Moreover, the role of the basal cells of the human prostatic epithelium is still uncertain. These cells have been described as undifferentiated cells, precursors of luminal cells, reserve and myoepithelial cells. The objective of the present study was to assess the similarities and/or differences between the epithelium of the human prostate and that of other laboratory animals and thus derive information about the potential functions of basal cells in the human prostate. In the human, basal cells form a continuous layer of cells resting on the basement membrane and upon which rests a layer of luminal cells. This results in a stratified columnar epithelium of two layers of cells, unlike the sporadic appearance of basal cells observed in other species where it results in a pseudostratified epithelium. In addition, the ratio of basal to luminal cells in the human is about 1:1, while the average ratio in the other animal species examined is about 1:7. Furthermore, the gap junctional proteins connexin 26 and 43, are present between basal and luminal cells in the human, thus suggesting that these cells communicate directly with each other. In addition, the ultrastructure of the human basal cells shows morphological evidence of differentiated but not of undifferentiated cells. Moreover, the presence of junction-like structures between adjacent basal cells suggests that these cells form a blood-prostate barrier. In this way, basal cells could prevent substances derived from the blood from directly coming in contact with the luminal cells. Human basal cells could thus regulate functions of the luminal cells by being part of a two-cell mechanism somewhat analogous to thecal and granulosa cells in the ovary.     

Liquid nitrogen-based quick freezing: Experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface

The limitations of chemical fixation in permitting the 1:1 quantitative correlations required for convincing ultrastructural explanations of cell biological processes are noted. We describe techniques for obtaining highly reproducible direct quick freezing on the polished surface of pure copper bars dipping into a static dewar of liquid N₂. The importance and the ease of testing and obtaining bounce suppression with commerically available equipment is emphasized. Artefacts caused by tissue damage and bad freezing are illustrated, and a hitherto unrecognized population of presynaptic membrane attached vesicles is described in Torpedine electric organ. Between 15 and 20% of the synaptic vesicles are attached to ca. 30% of the cytoplasmic face of the presynaptic terminal membrane. There is a close correlation between the occurrence of such attachments and the application of electrocyte basal lamina to the external face. We suggest that these vesicles are the ‘membrane operators,’ ‘vesigates,’ and ‘highly active subpopulation’ of vesicles whose existence has been invoked to explain biochemical data in other laboratories. We further speculate that relatively selective Ca pumping by this immediately submembranous population leads to displacement of acetylcholine (ACh) and reloading with newly synthesized ACh. The preferential release of the latter would then be expected.     

Comparison of osmium/sonication and edta/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique has a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.     

Cornea and its adaptation to environment and accommodation function in veiled chameleon (Chamaeleo calyptratus): ultrastructure and 3D transmission electron tomography

We investigate the ultrastructural features and 3D electron tomography of chameleon (Chamaeleon calyptratus) which is a native of desert environments of Saudi Arabia. The corneas of the chameleon were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for electron microscopy and tomography, and observed under a JEOL 1400 transmission electron microscope. The thin cornea (21.92 μm) contained 28–30 collagen fibril lamellae. The middle stromal lamellae (from 13 to 19) contained keratocytes with a long cell process and filled with granular material. The CF diameter increased from lamella 1 (30.44 ± 1.03) to Lamella 5 (52.83 ± 2.00) then decreased towards the posterior stoma. The percentage of large CF diameters (55–65 nm) was very high in the lamellae L14 (38.8%) and L15 (85.7%). The mean PGs area of the posterior stroma (448.21 ± 24.84 nm²) was significantly larger than the mean PGs area of the anterior, (309.86 ± 8.2 nm²) and middle stroma 245.94 ± 8.28 nm²). 3D electron tomography showed the distribution of PGs around and over the CF. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. This accommodation function is achieved by bending of the cornea. During bending the anterior stroma was stretched and the posterior stroma was compressed due to the presence of small CFs. The middle stroma remained stiff due to the presence of large CFs. Large proteoglycans not only maintain hydration for a longer period of time, but also act as a lubricant to neutralise the shear forces in the anterior and posterior stroma during bending.     

Morphometric characteristics of choroid plexus epithelial cells in cases with significantly different psammoma bodies' presence

Objectives: Taking into the consideration the fact that psammoma bodies have never been observed in stroma of any other organ as aging change, the aim of our research was to prove some structural similarities of choroids plexus and tumors psammoma bodies, and their possible connection with choroids plexus epithelial cells atrophy. Materials and Methods: We used 30 cadavers' right lateral ventricle central parts of choroid plexus as material. Tissue samples were routinely processed for the applied histochemical and immunohistochemical stainings. ImageJ software was used for morphometric analysis. Results: Cluster analysis showed the presence of two significantly different groups. The first group included the cases with sparse psammoma bodies and milder epithlelial atrophy with dome cells and vacuoles presence in older cases. The second group included the cases with numerous psammoma bodies and more severe epithelial atrophy, significant cystic formations and epithelial flattening presence, even in younger cases. Immunohistochemical analysis showed positive reaction of psammoma bodies and choroid plexus stroma on S100 protein. Application of S100 A8/A9 marker showed partial positive psammoma bodies' reaction and significant presence of S100 A8/A9 positive cells in choroid plexus blood vessels and stroma, especially, in cases with psammoma bodies' positive reaction on this marker. Conclusions: So, presence of more numerous psammoma bodies' might be associated with more severe choroids plexus epithelial cells atrophy. Immunohistochemical analysis showed psammoma bodies' positive reaction on S100 protein and the presence of S100 A8/A9 positive cells in stroma of cases with psammoma bodies' positive reaction on this marker. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Confocal microscopy of the in-situ crystalline lens

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.     

Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.     

Three-dimensional reconstruction of colon carcinoma metastases in liver

Resection of liver metastases in patients with colon cancer increases survival but success depends on removal of all tumour tissue. For this purpose, understanding of spatial relationships between metastases and liver architecture is essential. Because metastatic cancer growth is essentially a three-dimensional (3D) event, we decided to apply 3D reconstruction techniques to study these spatial relationships between metastases and liver structures such as blood vessels, stroma and the liver capsule (Glisson’s capsule). Colon carcinoma metastases were experimentally induced in rat liver by injection of colon cancer cells (CC531) into the portal vein. Three weeks later, livers from these animals and control livers were removed and immediately frozen in liquid nitrogen. Thirty-seven to 110 consecutive sections were used for each 3D reconstruction of 26 metastases in eight livers. Contours of different structures were stained by (immuno)histochemical means, traced in each section and stored in a database. From the contour model, a volume model was generated. Among the 26 metastases, seven were found to grow distantly from the liver capsule. They were small and consisted of well-differentiated cancer cells that were totally surrounded by a basement membrane and stroma which was always connected with adjacent blood vessels of a portal tract. The remaining 19 metastases showed a more advanced pattern of development. Infiltration of poorly differentiated colon cancer cells progressed through the stroma at various sites and areas of direct contact between cancer cells and hepatocytes were frequently found. This type of outgrowth of cancer cells was only found when metastases had made contact with the liver capsule. However, some areas in sections of these advanced stages still resembled small metastases. On the basis of these findings, we conclude that stroma affects the differentiation pattern of cancer cells and has at least a dual role in tumour growth. On the one hand it limits invasion of cancer cells in the surrounding host tissue. On the other hand, stroma formation at the capsule, which consists mainly of granulation tissue, facilitates outgrowth of the tumours. Furthermore, our 3D reconstructions demonstrate the spatial heterogeneity of larger metastases and the importance of a 3D approach to understand growth and development of metastases in general and colon cancer metastases in the liver in particular.     

Morphology,distribution, and abundance of antennal sensilla of male and female macropterous and brachypterous small brown planthopper,Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae)

The antennal sensilla of both genders of macropterous and brachypterous adults of the small brown planthopper, Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) were examined using light and scanning electron microscopy. Scanning electron microscopy revealed seven types of antennal sensilla in adult L. striatellus which were not evenly distributed on all antennal segments. Sensilla chaetica, a sensillum campaniformium and a Böhm bristle were found on the scape. Sensilla chaetica, sensilla trichodea, sensilla placodea which always present as plaque organs, sensilla basiconica and a sensillum campaniformium were present on the pedicel. Three sensilla basiconica and one sensillum coeloconicum containing two sensory pegs were located on the swollen sensory region of the basal flagellum. Pores observed on the surface of s. trichodea and s. placodea suggest these organs probably play a role in olfaction, whereas the aporous s. chaetica with flexible sockets probably function as mechanoreceptors. The aporous s. basiconica with inflexible sockets are probable to be thermo‐hygroreceptors while the Böhm bristle and s. campaniformia may act as antennal proprioceptors. The function of s. coeloconicum remains uncertain. The numerical dominance of antennal olfactory receptors suggests olfaction is an important function of the antenna in L. striatellus. Although a small degree of sexual/wing dimorphism was observed in the numbers of sensilla and in the length and width of antennae and antennal segments, the basic shape and structure of the antennae and antennal sensilla did not differ between the gender or wing form in L. striatellus. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Histology and ultrastructure of Aedes albopictus larval midgut infected with Bacillus thuringiensis var. israelensis

Histological and ultrastrucutural alterations in the midgut of Aedes albopictus larvae infected with Bacillus thuringiensis var. israelensis (Bti) were observed by light and transmission electron microscopy (TEM). Two formulations of Bti were used: granulated and powder, with 0.2% active ingredient in 90 larvae of Ae. albopictus distributed in three containers containing 30 larvae each (one control group and two test groups). The midgut epithelium of the control group presented flattened and elongated cells with mace-shape with a narrow base. Midgut epithelium cells' surface was convex and had a large circular nucleus located in the median-apical portion of the cell. These cells also presented a basal lamina with a small accumulation of extracellular fibrous matrix, thus characterizing a basal membrane, with a muscle layer and a peritoneal membrane more externally. After Bti ingestion, the larvae stopped/slowed their natural movements down in 5 min. After 30 min approximately, the swimming movements stopped completely. Internally, the intestinal cells showed a disorganization of the basal processes, dilatation and fragmentation of the rough endoplasmic reticulum, with intense cytoplasmic vacuolization. There were concentric dense laminas accumulated in the cytoplasm, and these residual membranous bodies were seen greatly increased in size after 60 min. Mitochondria, fragments of rough endoplasmic reticulum and other remainder organelles were surrounded and segregated from the cytoplasm by exocytosis. This article reports the histopathological alterations in the midgut of Ae. albopictus after infection with Bti and contributes to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against mosquito larvae.     

The effect of six fixatives on the areas of rabbit neurons and rabbit and rat cerebral slices.

Cell bodies of cerebral neurons from rabbits were isolated by hand, transferred to a microscope slide in a ‘199’ medium, and the projected areas of their cytoplasm and nuclei were measured. In sixty-four cells there was a strong correlation between the projected areas of the cytoplasm and the nuclei (r=0.66, P < 0001), and the ratio of the projected areas was 11.6. The medium was then replaced by the following fixatives: formalin (10% v/v), Bouin's, Carnoy's, Susas, glutaraldehyde (5% v/v), and osmium tetroxide (1% w/v). Cerebral slices were obtained from the grey and white matter of rabbit and rat, and were also measured before and after treatment with similar fixatives. Relative to the unfixed areas, glutaraldehyde and osmium tetroxide had no significant effect on the projected areas of isolated cells, Carnoy's fixative shrunk the areas of the cytoplasm by means of 16.26%, Bouin's by a mean of 49%, and Susas by a mean of 65%. The shrinkage of the cytoplasm and the nuclei was not significantly different from that of the nuclei for each of these three fixatives individually, but with formalin the mean shrinkage of the cytoplasm was 46% while the nuclei did not shrink significantly. Using the same fixatives the effect on the areas of the cerebral slides from rabbit and rat were as follows: glutaraldehyde and osmium tetroxide caused no change in area; Carnoy's, formalin and Bouin's fixative diminished the areas by a mean of 10–20%, and Susa's by a mean of 35%. It was concluded that a particular fixative often caused a different degree of shrinkage to the cytoplasm, nuclei and cerebral slice. In general, the lower the osmotic pressure of the fixative, the less shrinkage it induced.     

Convergence in dental histology between the late Triassic semionotiform Sargodon tomicus (Neopterygii) and a late cretaceous (Turonian) pycnodontid (Neopterygii: Pycnodontiformes) species

Microstructural scanning electron microscope investigation was performed on sectioned and surface‐etched isolated, prehensile teeth of the Late Triassic semionotiform species Sargodon tomicus and Pycnodontidae incertae sedis from the Late Cretaceous. The teeth of both taxa display a system of vascular canals penetrating the dentine and the overlying hypermineralized acrodin cap; small tubules are radiating at an angle to the long axis of the canals, interpreted as residual spaces left by odontoblast cell processes. This is the first detailed account of vascular acrodin encountered in a pycnodont species. New information is revealed also about Sargodon dental histology in the shape of mineralized remnants of the basal lamina at the acrodin–dentine junction. This implies that deposition of the acrodin organic matrix proceeded centrifugally by the cells of the inner dental epithelium, probably with minor collagen contribution from odontoblasts. This is contrary to the more typical centripetal formation (beneath the basal lamina) of the acrodin layer implied for the studied pycnodontid teeth. The rare occurrences of vascular acrodin within Actinopterygii, and the demonstrated differences in its histogenesis, do not suggest the usefulness of the tissue as systematic character but rather point to its adaptive significance. The superficial increase in the order of acrodin bundle orientation, observed in both species, is similarly regarded as convergently acquired mechanical adaptation. The observed uneven shape of crystallite rows and lesser degree of mineralization of the inner collariform ganoin, compared to its outer portion, is indicative of epithelial‐ectomesenchymal interaction and qualifies the tissue as enameloid. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy

Three-dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide-adenine dinucleotide, NADH, and reduced nicotinamide-adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 μm thickness of the cornea. Two-photon excitation scanning laser microscopy with near-infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 μm within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two-photon microscopy for future redox fluorescence imaging of cellular metabolic states.     

Comparative histochemical analysis of glycoconjugates in the nasal vestibule of camel and sheep

While Corriedale sheep survive in a wide range of climates, which prevents them to specialize for one climatic condition only, dromedary camels strictly adapted to desert areas. This demands more adaptive mechanisms to hot, dry conditions in camels than in sheep. Being the entrance of the nasal cavity, nasal vestibule is subjected to various environmental stressors. A protective way is the lining epithelium which is cornified in camel, but not in sheep. Mucus nasal secretions also play a key role in the protection of underlyings. Additionally, arterio‐venous anastomosis is present in the lamina propria of the nasal vestibule of camel. In the present paper, sugar residues in the nasal vestibule of camel were analyzed and compared with those of sheep using 14 types of lectins to explore the distribution of glycoconjugates that may help the function of camel nasal vestibule in desert environment. In camel, none of the lectins could label the basal cells of the vestibular epithelium, although the basal cells reacted with six lectins in sheep. In camel, LEL and RCA‐120 markedly labeled the luminal surface. WGA, DBA, SBA, and VVA produced marked intensities on the luminal surface in sheep. The mucous glands reacted with six lectins: WGA, s‐WGA, VVA, PNA, PHA‐E, and PHA‐L in camel, while all lectins used except s‐WGA and PHA‐E reacted in the sheep. In summary, great differences are observed in the glycoconjugate expression between camel and sheep. This suggests that these glycoconjugate are related to camel's tolerance for environmental stressors.     

Preservation and visualization of the sea urchin embryo blastocoelic extracellular matrix.

Several methods were utilized to visualize the structure and orientation of the blastocoelic extracellular matrix (ECM) in Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Rapid freezing in liquid propane cooled to LN2 temperatures followed by freeze substitution was used to preserve the ECM without shrinkage due to dehydration. Scanning, transmission, and light microscopy were employed to elucidate the ECMs' structure. The blastocoelic ECM consisted of parallel fibrillar sheets that were interconnected by finer filaments and oriented along the animal-vegetal axis. The ECM completely filled the blastocoelic cavity as viewed by scanning electron microscopy. The basal lamina could be distinguished from the blastocoelic ECM as a thin coat on the plasma membrane of epithelial cells; the ECM was in contact with this coat. In contrast, the blastocoelic ECM attached directly to the plasma membrane of primary mesenchyme cells (PMC) which did not possess a basal lamina. The blastocoelic ECM was isolated as an intact "bag" and probed in a hydrated state with Con A and alcian blue. Confocal microscopy confirmed that the entire blastocoel was filled with a fibrillar ECM. These approaches offer advantages for future studies of the ECMs of sea urchin embryos and their roles in gastrulation.     

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.     

Real-time confocal microscopy of keratocyte activity in wound healing after cryoablation in rabbit corneas

A modified tandem scanning confocal microscope was used for real-time in vivo examination of the rabbit cornea following a cryogenic injury. The corneas of New Zealand white rabbits were frozen with aprobe that had been cooled by immersion in liquid nitrogen, effectively destroying keratocytes in a central 5 mm diameter zone throughout the total thickness of the cornea. In these eyes, keratocyte repopulation and corneal stromal wound healing proceeded similarly to that which occurs after epikeratophakia, a refractive surgical procedure designed to change the curvature and optical power of the cornea. In epikeratophakia, a cryolathed donor corneal stroma lenticule is sutured onto the bare stroma of the recipient cornea. The collagen tissue lenticule is repopulated by keratocytes (corneal fibroblasts) that migrate in from the host cornea. In our study, the confocal microscope permitted sequential, noninvasive examination of the corneal stroma in the treated animals. Necrosis of the keratocytes, followed by activation of the remaining viable cells in the corneal periphery, was observed in the first 2 to 3 days after cryo injury. A fine stromal fibrous network was seen to develop; in three eyes, this network progressed to the development of a retrocorneal fibrous membrane and dense stromal fibrosis, both of which resulted in significant loss of corneal clarity. Our results suggest that the confocal microscope may be a valuable tool to provide much needed information on wound healing processes at the cellular level after corneal surgery and injury.