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PubMed2001

Visualization of negative signaling in B cells by quantitative confocal microscopy.

Numerous biochemical experiments have invoked a model in which B-cell antigen receptor (BCR)-Fc receptor for immunoglobulin (Ig) G (FcgammaRII) coclustering provides a dominant negative signal that blocks B-cell activation. Here, we tested this model using quantitative confocal microscopic techniques applied to ex vivo splenic B cells. We found that FcgammaRII and BCR colocalized with intact anti-Ig and that the SH2 domain-containing inositol 5'-phosphatase (SHIP) was recruited to the same site. Colocalization of BCR and SHIP was inefficient in FcgammaRII-/- but not gamma chain-/- splenic B cells. We also examined the subcellular location of a variety of enzymes and adapter proteins involved in signal transduction. Several proteins (CD19, CD22, SHP-1, and Dok) and a lipid raft marker were co-recruited to the BCR, regardless of the presence or absence of FcgammaRII and SHIP. Other proteins (Btk, Vav, Rac, and F-actin) displayed reduced colocalization with BCR in the presence of FcgammaRII and SHIP. Colocalization of BCR and F-actin required phosphatidylinositol (PtdIns) 3-kinase and was inhibited by SHIP, because the block in BCR/F-actin colocalization was not seen in B cells of SHIP-/- animals. Furthermore, BCR internalization was inhibited with intact anti-Ig stimulation or by expression of a dominant-negative mutant form of Rac. From these results, we propose that SHIP recruitment to BCR/FcgammaRII and the resulting hydrolysis of PtdIns-3,4,5-trisphosphate prevents the appropriate spatial redistribution and activation of enzymes distal to PtdIns 3-kinase, including those that promote Rac activation, actin polymerization, and receptor internalization.

Actins↗
PubMed1993

Improvements for the anatomical characterization of insect neurons in whole mount: the use of cyanine-derived fluorophores and laser scanning confocal microscopy.

The optical sectioning capability of the laser scanning confocal microscope was utilized to image dye-filled neurons within whole-mounted insect ganglia. Specific pterothoracic interneurons, in the moth Manduca sexta, were retrogradely filled with Neurobiotin and subsequently visualized with a monoclonal anti-biotin conjugated with one of the following fluorophores: fluorescein, and the newly developed cyanines, Cy3.18 (Cy3) and Cy5.18 (Cy5). Overall, the Cy5 fluorophore was best suited for imaging insect neurons within ganglia. This new methodology allowed us to identify and characterize morphologically a collection of descending multisegmental interneurons with large or small diameter somata. A variety of larger molecular weight (10,000 daltons) tracers was also used to examine the possibility of nonselective filling of neurons with Neurobiotin, possibly through gap junctions. We also investigated the usefulness of Cy3 and Cy5 as fluorophores for transmitter immunostaining of neurons in whole mount. Neurons immunoreactive for serotonin and the neuropeptides, FMRFamide and SCPB, were imaged in the brain and the pterothoracic ganglion. The central projections of some of these immunoreactive neurons were imaged in their entirety.

Animals↗
PubMed2002

Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy.

Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning microscope. Rat acini and ducts were loaded with various Ca2+ sensing fluorophores (Fluo-4, Fura-Red, Calcium Green-1, Indo-1 and Fura-2). Only Fura-2 loaded evenly into acinar clusters and ducts and UV laser excitation at 351 and 364 nm gave signals showing opposite sensitivity to Ca2+ concentration changes. About 15% of acini or single cells within acini responded to ATP or UTP, yet all responded to carbachol. In contrast, all ducts responded to ATP/UTP. ATP stimulated uptake of a fluorescent anion, luciferin, but again only in a few acinar cells. RT-PCR analysis on acini samples collected from whole pancreas suspensions revealed transcripts for P2Y(2), P2Y(4) and P2X(1), P2X(4) receptors. The low number of functional P2 receptors in acini might be related to the fact that they release ATP. Thereby acini would avoid autocrine stimulation and initiation of autodigestive processes, such as occurs in pancreatitis.

Adenosine Triphosphate↗
PubMed1998

Changes in morphology and behaviour of retinal growth cones before and after crossing the midline of the mouse chiasm - a confocal microscopy study.

The growth of retinal axons was investigated in different regions of the optic chiasm in C57 pigmented mouse embryos aged embryonic day 13 (E13) to E15. Individual retinal axons and their growth cones were labelled anterogradely by DiI and imaged using a confocal imaging system. In aldehyde-fixed embryos, retinal growth cones display a simple form in the optic nerve and become more complex in morphology in the chiasm. The complex form is particularly prominent in those axons that turn to the ipsilateral tract in the premidline region of chiasm. Moreover, complex growth cones are also commonly found in axons in the postmidline chiasm, which are markedly different in morphology from those axons in the premidline region, suggesting that the postmidline chiasm contains a novel environment for the pathfinding of retinal axons. In another experiment, the dynamic growth of retinal axons is studied in a brain slice preparation of the living retinofugal pathway. Retinal axons show an intermittent growth across the premidline and postmidline chiasm. Extensive remodelling of growth cone form followed by a shift in growth direction is commonly seen during the pause periods, indicating that signals that guide axon growth across the chiasm are not restricted to the midline, but are laid down throughout the chiasm. Moreover, dramatic changes in axon trajectory are noted first at the premidline chiasm where the uncrossed axons segregate from the crossed axons, and second at the postmidline chiasm where specific sorting of retinal axons according to their position in the dorsal ventral retinal axis and their ages are known to take place. These results show that there are two distinct environments, separated by the midline in the chiasm, where axons show different responses to local guidance cues and develop the distinct fibre orders.

Animals↗
PubMed1997

Calcium signals from intact rabbit ciliary epithelium observed with confocal microscopy.

PURPOSE: We investigated patterns of evoked calcium signals to learn about the function of the calcium second messenger system in ciliary epithelium. METHODS: Isolated infact ciliary processes were loaded with fluo-3/AM and observed with a Bio-Rad MRC-600 laser scanning confocal imaging system, before, during, and after perfusion with catecholamines, cholinergic agents, and autocoids. RESULTS: One microM acetylcholine (ACH) and 10 microM carbachol (CARB) induced an atropine-sensitive increase in intracellular free calcium ion concentration ([Ca2+]i), considerably greater in NPE than in PE. 10 microM epinephrine (EPI) and 100 microM phenylephrine (PHE) increased [Ca2+]i in NPE and PE, in this case PE > NPE. These effects were blocked by prazosin. 10 mM caffeine (CAF) increased of [Ca2+]i in NPE and PE (NPE > PE) and sometimes produced very slow oscillations with an interval of 10 to 25 s. Prior administration of CAF strongly suppressed the effects of ACH, CARB, EPI, PHE, histamine, and adenosine 5-triphosphate (ATP). One hundred microM ryanodine (RYA) or thapsigargin (TG) increased [Ca2+]i in NPE and PE (NPE > PE). CONCLUSIONS: In the ciliary epithelium: (1) different patterns of evoked transients and oscillations of calcium were seen in response to agonists; (2) NPE appeared to contain a predominance of muscarinic receptors, while the PE is dominated by alpha 1-adrenergic receptors and (3) the increase in [Ca2+]i by CAF or RYA or TG in either cell layer and the blocking effects of these agents upon agonist, evoking increases in [Ca2+]i, suggested involvement of both the cyclic adenosine diphosphate ribose and the inositol 1, 4, 5 triphosphate (InsP3) systems in the regulation of intracellular calcium.

Aniline Compounds↗
PubMed2001

A better potassium hydroxide preparation? In vivo diagnosis of tinea with confocal microscopy.

BACKGROUND: Traditional diagnostic testing for dermatophyte infection currently requires skin scraping for light microscopy and/or fungal culture or skin biopsy. Immunofluorescent microscopy can also be used with calcofluor stain. All of these tests can be time-consuming to perform, require a waiting period for results, and are invasive. We investigated the use of a real-time, noninvasive, confocal microscope in visualizing dermatophyte hyphae in vivo. OBSERVATIONS: Confocal microscopic imaging of active tinea can clearly identify dermatophyte hyphae within the upper epidermis after potassium hydroxide application. The hyphae appear as bright linear branching objects not found in uninvolved skin. CONCLUSIONS: It is possible to immediately and painlessly image dermatophyte hyphae in active lesions of tinea by means of a confocal microscope. With further improvement, imaging devices may be available to physicians to instantly and noninvasively evaluate a variety of skin disorders in microscopic detail.

Adult↗
PubMed2002

Imaging solute distribution in capillary electrochromatography with laser scanning confocal microscopy.

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.

Journal Article↗
PubMed1996

Transient corneal stromal and endothelial changes following soft contact lens wear: a study with confocal microscopy.

PURPOSE: Previous studies have described transient corneal endothelial changes in non-contact lens wearers after a short period of soft contact lens wear by means of contact and noncontact specular microscopy and modified slit lamp biomicroscopy, which provide magnifications from 60 to 100x. In this investigation, we documented and characterized these contact lens-related corneal changes using the white light, real-time confocal microscope, which is capable of cellular resolution imaging of all layers within the cornea at magnifications of 100 to 500x. METHODS: We used a clinical confocal microscope to study corneal changes in three patients wearing a high water content soft contact lens for the first time. RESULTS: In one patient, endothelial changes consisting of irregularly shaped, round or oval, dark regions were observed within the endothelial mosaic. Scattered hyper-reflective keratocyte nuclei were seen in the posterior stroma. The keratocytic and endothelial changes were most evident 20 minutes after placement of the lens. By 30 minutes, the changes were fewer and less prominent, and the brightness of the highly reflective keratocyte nuclei had decreased. CONCLUSIONS: These studies show, for the first time, that the transient changes associated with contact lens wear occur not only in the endothelium, but also in the corneal stroma. It has been suggested that the changes result from an increase in CO2 and lactic acid, which causes a transient reduction in the corneal pH. We hypothesize that the resulting acidic environment may induce gene expression that causes changes in the involved nuclei, which in the keratocytes become hyper-reflective, and in the endothelium become enlarged, resulting in posterior displacement of the cell membrane and producing the dark "blebs" and irregular lines observed at this level of the posterior cornea.

Contact Lenses, Hydrophilic↗
PubMed2005

Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy.

Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 +/- 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 +/- 2.8 microm. In addition, we could calculate that each osteocyte spans an average of 4180 +/- 673 microm3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 +/- 0.02 microm per 1 microm3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 +/- 0.03 microm2 per 1 microm3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% +/- 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 +/- 145 microm, 1509 +/- 113 microm2, and 394 +/- 49 microm3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.

Animals↗
PubMed1997

Cellular changes induced by chronic nitric oxide inhibition in intact rat basilar arteries revealed by confocal microscopy.

BACKGROUND: Cellular aspects of remodelling have not been investigated fully in intact vessels due to lack of appropriate methodology. OBJECTIVE: To determine the cellular alterations induced by chronic inhibition of nitric oxide (NO) production in intact rat basilar arteries by combined use of perfusion myography and a laser scanning confocal microscope. METHODS: Wistar-Kyoto rats were treated with 10 mg/kg per day NG-nitro-L-arginine methyl ester (L-NAME) for 3 weeks. Basilar arteries from treated and age-matched Wistar-Kyoto rat controls were mounted on a perfusion myograph, stained with the nuclear dye Hoechst 33342 and fixed under pressure. The segments were mounted on a slide and visualized using the 364 nm line of a laser scanning confocal microscope. MetaMorph software was used to obtain optical sections from the vessel and for morphology determinations. RESULTS: L-NAME treatment induced hypertension (systolic blood pressure control 129.2+/-2.7 mmHg and SBP L-NAME treatment 176.3+/-5.2 mmHg, P< 0.001). Compared with control rat arteries, arteries from treated rats had a reduced lumen diameter, similar wall thickness and an increased wall: lumen ratio. L-NAME treatment induced specific changes in adventitia, media and intima, namely an increase in number of adventitial cells and in adventitia thickness, a reduction in number of smooth muscle cells with no change in media thickness and reductions in number of endothelial cells, size of nuclei and luminal surface area. CONCLUSIONS: Hypertension induced by chronic inhibition of NO production is associated with eutrophic remodelling of rat basilar artery. However, within this overall maintenance of constant volume, there are marked cellular changes in adventitia, media and intima. The separate contributions of inhibition of NO production and hypertension to the remodelling process need to be elucidated.

Animals↗
PubMed2006

In vivo corneal confocal microscopy comparison of intralase femtosecond laser and mechanical microkeratome for laser in situ keratomileusis.

PURPOSE: To assess and compare corneal modifications induced by IntraLase PulsionFS femtosecond laser and mechanical microkeratome Hansatome for laser in situ keratomileusis (LASIK) using the new-generation Heidelberg Retina Tomograph II (HRT II)/Rostock Cornea Module confocal microscope. METHODS: In this case-control study, 24 eyes of 12 patients were examined with the HRT II in the follow-up of IntraLase femtosecond laser for LASIK myopic surgery. Twenty eyes of 10 patients were also examined after microkeratome Hansatome-LASIK surgery. In both groups, the patients underwent the first follow-up examination on day 7 and the last 12 months after surgery. Morphologic modifications of corneal architecture were evaluated, and comparisons were made between the two flap-formation techniques. RESULTS: Evaluation of both groups on day 7 showed keratocyte transformation, most likely related to cellular activation beneath the interface. The flap margin after the IntraLase technique appeared microscopically as a clear-cut edge that included the epithelial plug. At month 2, secondary fibrosis, adjacent to the still well-defined IntraLase flap edge, was observed. This reaction diminished with time, leaving a fibrotic scar adjacent to a wound constriction originating from the surrounding stroma. The flap margin of the mechanical microkeratome had the appearance of a less clearly identified fibrotic scar with no epithelial plug. CONCLUSIONS: This study reveals morphologic similarities between the interfaces obtained by femtosecond laser and mechanical microkeratome, probably because the same excimer laser performed the photoablation. However, the IntraLase flap margin showed greater fibrotic scarring than that induced by the mechanical microkeratome.

Adult↗
PubMed2006

FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in.

Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

Arrestins↗
PubMed2001

An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events.

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40--100 microm thickness of paraformaldehyde-fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope. This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples. The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.

Animals↗
PubMed2006

Quality assessment of confocal microscopy slide based systems: performance.

BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.

Animals↗
PubMed2000

Technical advance: an aniline blue staining procedure for confocal microscopy and 3D imaging of normal and perturbed cellular phenotypes in mature Arabidopsis embryos.

A new method is described for fluorescent imaging of mature Arabidopsis embryos that enables their cellular architecture to be visualized without the need for histological sectioning. Mature embryos are stained with aniline blue and cleared with chloral hydrate to allow high-resolution confocal imaging of individual cells within the embryo prior to germination. The technique allows the collection of longitudinal optical sections throughout the cotyledon, hypocotyl and root of wild-type Arabidopsis C24 embryos. Every cell within the mature embryo can be visualized with sufficient clarity and resolution to allow three-dimensional analysis of cellular architecture. Optical sectioning of mutant gnom, short-root and scarecrow embryos, and through root meristems disrupted as a consequence of targeted misexpression of diphtheria toxin, demonstrate the potential of this technique for visualizing the cellular organization of mutant and perturbed embryonic phenotypes.

Aniline Compounds↗
PubMed2005

Direct visualization of colloidal rod assembly by confocal microscopy.

The development of model materials and image processing methods to directly visualize and quantify colloidal rod assembly by means of confocal laser scanning microscopy (CLSM) is reported. Monodisperse fluorescent colloidal rods are prepared by the uniaxial extensional deformation of sterically stabilized microspheres at elevated temperatures. The particles are stably dispersed in refractive index matching mixed organic solvents for CLSM. An image processing algorithm is developed to detect rod backbones and extract particle centroids and orientation angles from the CLSM image volumes. By means of these methods we quantify the distribution of rod orientation angles in self-assembled structures of rods formed by sedimentation. We find the observations to be consistent with aspect-ratio-dependent jamming and orientational order/disorder transition in the rod sediments.

Journal Article↗
PubMed1997

[Confocal microscopy of the corneal after photorefractive keratectomy with the excimer laser].

BACKGROUND: In photorefractive keratectomy (PRK) procedures, a variable superficial central corneal scar formation ("haze") can be observed following removal of corneal stromal tissue. Today, a near to normal slit lamp finding is observed one year postoperatively in most patients. We employed the slit scanning confocal microscope to study the corneal morphology years after PRK. METHODS: We selected 5 patients, who had been subjected to unilateral photorefractive surgery 1-3 years earlier and who had no corneal haze upon slit lamp examination. As controls we investigated the non-treated corneas of these patients, 5 healthy controls and 5 contact lens wearers. The confocal microscopic investigation was performed with 25x, 40x and 50x water immersion objectives. The video signal was synconized with the slit scan and stored on S-VHS video tape. By reviewing the videos in the single frame mode, all corneal layers could be qualitatively evaluated. RESULTS: Some minor abnormalities were observed in the epithelium of all PRK-treated eyes. In the epithelial basal cell layer some round structures of about the size of a cell with high reflectivity were observed. These changes were only occasionally found in contact lens wearers, but not in non-treated or normal control eyes. Bowman's layer was absent in the PRK treated eyes, instead, a fine layer of collagen tissue of increased reflectivity was found. The subepithelial corneal nerve plexus was normal in all non-treated eyes, whereas in the PRK-treated corneas nerve shape and branching pattern were changed to quite an extent. In the anterior stroma the keratocyte nucleus patterns indicated an increased cell density and irregular spacing, whereas a normal keratocyte pattern was found in the deeper stromal layers. A significant finding was the observation of rod and needle shaped highly reflective structures, which were limited to the area of the excimer laser keratectomy with a predominance in the anterior stroma. These longitudinal structures themselves consisted of linearly arranged highly reflective granules, which sometimes also were found as isolated dots within keratocyte processes. In long term contact lens wearers a comparable granule type, however with a singular and scattered arrangement, was variably found in all corneal regions and layers. In normal controls none of these findings were present. In contact lens wearers and PRK patients with a contact lens history, the corneal endothelium showed some degree of polymegathisms but no other specific findings. DISCUSSION: Up to now, refractive surgery with the excimer laser has been reported to elicit no other stromal changes but a mild fibroblast activation with subsequent scar tissue formation. In clinically clear corneas after PRK, we have described a new type of stromal deposit observed 1-3 years after surgery. As acute wound healing responses might have been expected to have passed at this point, this highly reflective stromal deposit can be assumed to consist of linear keratocyte processes filled with some highly reflective (degenerative?) matter as well as a corresponding extracellular stromal deposit arranged parallel to the stromal collagen bundles. Possibly, these stromal deposits represent the result of an inflammatory or degenerative stromal response resulting in the formation of stromal lipofuscein deposits. Visual acuity was not impaired in the patients investigated in this study. As these stromal deposits appear to be persisting years after surgery and possibly are irreversible in nature, a long term effect on the corneal physiology and function should carefully be monitored.

Adult↗
PubMed2003

Morphology of corneal Basal epithelial cells by in vivo slit-scanning confocal microscopy.

PURPOSE: To determine corneal basal epithelial cell density and morphology in a group of normal corneas and corneas with various conditions. METHODS: The central corneas of a group of 20 normal patients and 27 other patients who either had refractive surgery or had documented corneal pathology were examined with an in vivo slit-scanning confocal microscope. For all these patients in whom a well-defined basal epithelial layer was visualized, the basal cell density, area, and number of sides per cell were determined using customized software. RESULTS: The average basal cell density was 5,274 +/- 575 cells/mm2, the average cell area was 192 +/- 19.6 microm2, and the average number of cell sides was 5.5 +/- 0.1 sides for the group of normal controls. There were no statistically significant differences between basal cell density, cell area, or number of sides between the normal group and any of the other groups in which there were four or more patients. Corneas examined from patients with Fuchs' endothelial dystrophy or bullous keratopathy had hyperreflective basal epithelial cell borders. The measurement of the number of cell sides is not reliable using software developed for endothelial cell counting. CONCLUSION: Corneal basal epithelial cell density and area were found to be remarkably consistent in the variety of corneal disorders that we examined. This finding is further evidence that this aspect of corneal epithelial cell differentiation and maturation is tightly controlled with little variability between individuals even if a corneal condition is present.

Adult↗