Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Second-harmonic imaging microscopy of normal human and keratoconus cornea

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.     

Rabbit cornea microstructure response to changes in intraocular pressure visualized by using nonlinear optical microscopy

PURPOSE: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM). METHODS: Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea. RESULTS: Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP. CONCLUSIONS: NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.     

The role of dermatopontin in the stromal organization of the cornea

PURPOSE: Dermatopontin (DPT) is an abundant component of the stromal extracellular matrix; however, its function in the cornea is poorly understood. This study was conducted to determine whether DPT has a direct role in corneal matrix organization by investigating the ultrastructure of Dpt-null (Dpt(-/-)) mouse corneas. METHODS: Conventional light microscopy was used to compare the corneal thickness of Dpt(-/-) mice with that of the wild type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analyzed using image analysis software to determine fibrillar volume, fibril diameter, and spacing. RESULTS: Light microscopy demonstrated that Dpt(-/-) corneas in 2-month-old mice showed a 24% reduction in average stromal thickness compared with wild type (P < 0.001). The epithelium and Descemet's membrane appeared normal. Examination of Dpt(-/-) stroma by transmission electron microscopy indicated significant disruption of fibril spacing within the posterior lamellae, whereas the mid and anterior regions appeared largely unaffected compared with wild type. The collagen fibrils in Dpt(-/-) stroma showed a lower fibril volume fraction and a pronounced change in posterior fibrillar organization. There was no apparent difference in fibril diameter between Dpt(-/-) and wild-type mice. CONCLUSIONS: Collectively, these data suggest that DPT plays a key role in collagen fibril organization. The defects in collagen organization in Dpt(-/-) cornea appear to be most severe in the posterior stroma. It is possible that DPT interacts with corneal proteoglycans and that this interaction is involved in the maintenance of stromal architecture.     

Transparency, swelling and scarring in the corneal stroma

PURPOSE: This paper briefly reviews current explanations for corneal transparency and uses a well-developed model to try to explain the increased light scattering either accompanying corneal swelling or following phototherapeutic keratectomy (PTK). METHODS: The direct summation of fields (DSF) method was used to compute light transmission as a function of wavelength. The method requires input of a number of structural parameters. Some of these were obtained from electron micrographs and others were calculated from X-ray diffraction data. RESULTS: By swelling sections of stroma cut from different depths in the tissue, we have shown that fluid entering the cornea causes more swelling in the posterior lamellae than in the anterior lamellae. Furthermore, posterior lamellae can reach a higher final hydration than anterior lamellae. Collagen-free regions ('lakes') exist in corneas swollen in vitro and in Fuch's dystrophy corneas, many of which may be caused by the death of cells. The DSF method shows that local fibril disordering, increased refractive index mismatch, and increased corneal thickness together can account for a 20% increase in light scattering in a Fuch's dystrophy cornea at H=5.8 compared to the normal cornea. Additional scattering is probably caused by 'lakes'. The DSF method applied to PTK rabbit stroma with high levels of haze suggests that the newly deposited collagen is not the cause of the increased light scattering. CONCLUSIONS: Fluid is not uniformly distributed within the corneal stroma when the cornea swells. Increased hydration of posterior lamellae may be because of known differences in the glycosaminoglycans between the anterior and posterior stroma. Lamellar interweave in the anterior stroma probably limits the extent to which the constituent lamellae can swell. The DSF method can be used to account for increased light scattering in oedematous corneas but cannot account for haze following PTK.     

Ultrastructural analyses of enzyme-treated microfibrils in rabbit corneal stroma

Microfibrils have been identified within and between corneal collagen lamellae in a number of vertebrate species in a variety of developmental and pathological conditions, but they are relatively rare in normal adult animals. The present study was undertaken to analyze corneal microfibrils in adult rabbits using enzymatic digestion techniques. Transmission electron microscopy (TEM) showed clusters of 10-15 nm microfibrils arranged in quasi-parallel bundles within or between orthogonally arranged stromal collagen lamellae. When corneas were fixed with tannic acid/glutaraldehyde, the entire stroma showed increased electron density and microfibrillar bundles were heterogeneously stained. Peripheral fibrils were more electron-dense than those located more centrally. Following sequential detergent solubilization of unfixed corneas, all cellular elements were removed and collagen lamellae were distorted. Microfibrillar bundles remained intact, however, and resembled untreated controls. Subsequent treatment with pepsin, trypsin or elastase resulted in swollen corneal tissues in which collagen lamellae were no longer distinguishable but individual collagen fibrils maintained their morphological integrity. In these tissues microfibrillar bundles were rarely identifiable and were reduced to randomly oriented fragments or clusters of filamentous material. Testicular hyaluronidase or chondroitinase ABC did not affect the fibrils. These data indicate that rabbit corneal microfibrils are proteinaceous and that the tannic acid-staining component of the bundles is not glycosaminoglycan. The fibrils are indistinguishable from those identified as oxytalan in cornea and other ocular tissues. Moreover, their sensitivity to elastase and preferential staining with tannic acid/glutaraldehyde strongly suggest they may be related to the elastic system of fibrils.     

Quantitative analysis of collagen fiber in keratoconus

In order to identify the direct pathogenic factors involved in the stromal thinning of keratoconus, quantitative analysis of keratocytes, collagen fibers and collagen lamellae in keratoconus cornea was performed histologically by light and electron microscopy. Both normal and keratoconus corneas showed a similar cell density of keratocytes in the central stroma, therefore the total number of keratocytes in keratoconus cornea might be smaller than that of controls, because of the thinning of stroma in the keratoconus. The collagen lamellae in keratoconus corneas showed a significant decrease in number compared with controls. There was a direct relationship between the stromal thickness and number of collagen lamellae. On the other hand, there was no statistical significance between normal and keratoconus corneas in terms of the thickness of collagen lamellae. These results suggest that the thinning of the cornea in keratoconus might occur as the result of a defect of some collagen lamellae due to a disorganization during the process of collagen lamellae formation.     

Iron deposits in cornea in confocal microscope

PURPOSE: The study aimed to evaluate the iron deposits in corneas in confocal microscope. MATERIAL AND METHODS: The material comprised 16 eyes which underwent photorefractive keratectomy (PRK) procedure. The structure of corneas was evaluated between 3-10 years after PRK. The visual acuity after PRK was the same as the best corrected visual acuity before the procedure. The structure of corneas was evaluated in vivo using scanning slit confocal microscopy. The confocal images of corneas in patients after PRK were compared with confocal corneal images of patients with corneal scars (2 eyes), keratoconus (2 eyes), after radial keratotomy (RK) (2 eyes) and healthy patients. RESULTS: Within the central part of corneal epithelium and anterior part of stroma, the clusters of iron deposits were observed. They were round and produced different shapes. In the paracentral and peripheral part of corneas the subepithelial nerve plexus was detected. Beneath, the pattern of keratocytic nuclei, characteristic for state after PRK, was detected. In patients with corneal scars, keratoconus and after RK, the same clusters of deposits were detected. In cases of corneal scars, additionally high reflectivity of corneal structure was observed. CONCLUSIONS: The iron deposits in corneal structure arise in epithelium and anterior part of corneal stroma. The iron deposits which produce different shapes have no influence on visual acuity.     

Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. METHODS: Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice. CONCLUSIONS: The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.     

Assessment of the apparent intra- and inter-sample variability in the collagen fibril diameter in the posterior corneal stroma of rabbits. A transmission electron microscopy study

PURPOSE: To assess the natural variability in the diameter of small collagen fibrils in the lamellae of corneal stroma of healthy young rabbits. MATERIALS AND METHODS: The corneas of 6 young adult female New Zealand White rabbits (2.1 kg) were prepared for transmission electron microscopy (TEM) by fixation with a cacodylate-buffered 2% glutaraldehyde (pH 7.2-7.4, 320-340 mosm/kg). The corneas were embedded in Epon-Araldite, thin sections prepared from the central region of the posterior corneal stroma and stained with acidic uranyl acetate followed by acidic lead citrate. High magnification (x 33,000) micrographs were taken, and fibril diameters (FDs) measured at a final magnification of x 275,000 to a resolution of 2 nm. RESULTS: Assessment of sampling-related errors indicated that the average diameter of the fibrils within any particular micrograph could be estimated to within 1% or better by measures of 100 fibrils. Assessments of the intra-sample variance (6 micrographs taken from the same cornea) indicated a group mean FD of 32.4 +/- 3.6 nm, whilst the inter-sample variance (6 micrographs taken from 6 different corneas) yielded an average of 33.1 +/- 4.5 nm (n = 100 fibrils/micrograph, +/-SD). However, group-averaged data sets of FDs, while unimodal, were not normally distributed, and cumulative averaging indicated a fixed range of FDs across the data sets. CONCLUSIONS: Intra- and inter-sample variability in collagen FDs is very similar, but the analysis indicates that the collagen fibrils are not homogeneous and that closely adjacent lamellae can have subtle differences in average FD.     

An x-ray diffraction study of corneal structure in mimecan-deficient mice

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.     

Preliminary in vitro study of the histological effects of low fluence 193-nm excimer laser irradiation of corneal tissue

PURPOSE: To determine if moderate numbers of low fluence, 193-nm excimer laser pulses modify or damage the corneal stroma. METHODS: The corneal epithelium of fresh bovine eyes was scraped off and the exposed stroma was irradiated with 200 low fluence laser pulses from an argon fluoride excimer laser. This process was performed on five eyes each at two laser fluences, 10 mJ/cm2 and 30 mJ/cm2. The ten irradiated and three control (unirradiated) corneas were sectioned and studied by electron microscopy. The maximum and minimum thickness of the anterior layer of randomly oriented collagen fibers was measured using electron microscopy. RESULTS: The mean maximum thickness of the anterior randomly oriented layer of collagen was 1.23 +/- 0.45 microm in the control corneas, 0.67 +/- 0.32 microm in the corneas irradiated at 10 mJ/cm2, and 0.10 +/- 0.12 microm in the corneas irradiated at 30 mJ/cm2. The mean thickness of corneal stroma removed was 0.7 microm at a fluence of 10 mJ/cm2 and 1.1 microm at a fluence of 30 mJ/cm2. A thin, electron-dense pseudomembrane was present at both fluences. CONCLUSION: We report removal of bovine corneal stroma at 10 mJ/cm2--below the previously reported ablation threshold of 20 mJ/cm2.     

The structure and swelling of corneal scar tissue in penetrating full-thickness wounds

OBJECTIVE: The purpose of this study was to determine corneal thickness and to examine the collagen and proteoglycans in full-thickness corneal scars up to 16 months following wounding. METHODS: Ultrasound pachymetry was used to measure the depths of penetrating central scars and their surrounding areas in 16 rabbit corneas. Measurements were taken at regular intervals: 5, 10, 12, and 16 months after healing. Transmission electron microscopy was then used to study the stroma of the resulting scars to observe the collagen organization and the amount, as well as the size, of cuprolinic blue-stained proteoglycan filaments within the stroma. Furthermore, ex vivo swelling of selected wounded contralateral excised corneas was undertaken by the measured addition of distilled water. RESULTS: In vivo, the thickness of the scar tissue was significantly less than that of the surrounding tissue throughout the period studied. By 12 months the proteoglycan filaments within the scar were of a similar size and number to those within the adjacent tissue, whereas the collagen fibrils within the scar were still disorganized (collagen interweaving, lack of a lamellar structure). Once excised and allowed to swell in water, scar tissue thickness remained relatively unchanged, whereas the surrounding tissue swelled considerably. CONCLUSION: Disorganized fibril arrangement inhibits the normal swelling of the scar tissue, which remains reduced. Furthermore, even after many months of healing, collagen remodeling in corneal scar tissue is not complete.     

The specific architecture of the anterior stroma accounts for maintenance of corneal curvature

AIM: To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability. METHODS: Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer. RESULTS: After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states. CONCLUSIONS: The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.     

Observations of banding patterns (Vogt striae) in keratoconus: a confocal microscopy study

PURPOSE: To investigate Vogt striae in keratoconus using confocal microscopy. METHODS: The central cornea of 51 eyes of 29 subjects with keratoconus was observed using a slit-lamp biomicroscope, slit-scanning confocal microscope (TOMEY Confoscan 1), and corneal topographer (EyeSys 2000). RESULTS: Alternating dark and light bands were seen in the stromal images of 23 eyes examined. The bands corresponded with the appearance of Vogt striae on slit-lamp biomicroscopy examination. Bands were found most commonly in the posterior stroma. Posterior bands varied in width, ran mainly in a nearly vertical direction, and appeared to run a straight course through individual image frames. Keratocyte nuclei were located in between the bands. Posterior keratocyte density was unaffected by the presence of bands. Nerve fibers appeared to run a straight course through the bands. When present, bands in the anterior stroma showed greater variability in width and direction within a single frame. Bands were only present in the anterior stroma in more severe levels of keratoconus. The difference in banding pattern noted between the anterior and posterior stroma parallels the known collagen fiber arrangement in the anterior and posterior stroma. CONCLUSIONS: The bands apparent on confocal microscopy of the stroma of the keratoconic cornea correspond with Vogt striae on slit-lamp microscopy. It appears that these bands (and hence Vogt striae) represent collagen lamellae under stress. The stress pattern appears to radiate from the center of the cone and is consistent with the direction of striae when viewed with the confocal microscope.     

Changes in collagen orientation and distribution in keratoconus corneas

PURPOSE: To map the collagen orientation and relative distribution of collagen fibrillar mass in keratoconus corneal buttons. METHODS: Structural analysis was performed by obtaining synchrotron x-ray scattering patterns across the samples at 0.25-mm intervals. The patterns were analyzed to produce two-dimensional maps of the orientation of the lamellae and of the distribution of total and preferentially aligned lamellae. RESULTS: Compared with normal corneas, in keratoconus the gross organization of the stromal lamellae was dramatically changed, and the collagen fibrillar mass was unevenly distributed, particularly around the presumed apex of the cone. CONCLUSIONS: The development of keratoconus involves a high degree of inter- and probably intralamellar displacement and slippage that leads to thinning of the central cornea and associated changes in corneal curvature. This slippage may be promoted by a loss of cohesive forces and mechanical failure in regions where lamellae bifurcate.     

In vivo confocal microscopy findings in a patient with posterior amorphous corneal dystrophy

A 21-year-old man, with bilateral posterior amorphous corneal dystrophy, was studied by biomicroscopy, corneal topography and in vivo confocal microscopy. The best-corrected visual acuity was 6/21 in the right eye and 6/6.9 in the left eye. Biomicroscopy revealed bilateral, asymmetric, sheet-like opacification at the deep posterior stromal layer. The corneal topography displayed asymmetric against-the-rule astigmatism in the right eye and prominent steepening at the inferior paracentral cornea in both eyes. In vivo confocal microscopy of the corneas demonstrated microfolds and hyper-reflective layer at the posterior stroma just adjacent to the endothelial layer. The epithelium, Bowman's membrane, anterior stroma and the endothelial layer were normal. In vivo confocal microscopy is useful in evaluating the corneal dystrophies.     

In vivo corneal confocal microscopy in marfan syndrome

PURPOSE: To examine the cornea of patients with Marfan syndrome in comparison with a control group by using the in vivo confocal microscope. METHODS: Twenty-four eyes of 12 patients with Marfan syndrome had their corneas examined using the in vivo confocal microscope Heidelberg Retina Tomograph (HRT) II/Rostock Cornea Module. The control group included 24 eyes of 12 subjects who had their corneas examined by the same in vivo confocal microscope. RESULTS: Epithelium and neural plexus examination did not show any difference between the 2 groups. Examination of the stroma showed no significant differences concerning the morphology and density of keratocytes. The extracellular matrix of 16 of the 24 eyes of the Marfan group was clearly visible and showed thin highly reflective interconnected lines between keratocytes. In the healthy eye group, reflective lines were observed in only 5 of the 24 eyes. The endothelium of 14 corneas of the Marfan group showed brightly reflective particles. In no cornea of the control group were such particles observed. CONCLUSIONS: Highly reflective extracellular matrix of the stroma and brightly reflective particles among the endothelial cells were the 2 main corneal findings observed by using in vivo corneal confocal microscopy in patients with Marfan syndrome compared with a control group. Further studies need to be made to confirm these findings and eventually find new criteria for Marfan syndrome from the examination of in vivo corneal confocal microscopy.     

White light confocal microscopy of preserved human corneas from an eye bank

PURPOSE: To verify the possibility of using a commercially available ophthalmologic white light confocal microscope for imaging optical sections of donated corneas preserved at 4 degrees C, especially for endothelial evaluation. METHODS: Sixteen corneas donated to the Warsaw Eye Bank but excluded from use in surgery for serological or morphologic reasons were examined using a ConfoScan 3 confocal scanning microscope (Nidek Technologies, Padova, Italy) and a Konan Eye Bank KeratoAnalyzer specular microscope (Konan Medical, Inc.; Hyogo, Japan). Images of corneal structures were obtained, including epithelium, corneal nerves, stroma, and endothelium. The endothelial cell density was calculated for both microscopes, and the results were compared. RESULTS: For images obtained with the specular microscope, mean (+/- SD) endothelial cell density was 2168.6 +/- 404.0 cells/mm; for confocal microscope images, mean +/- SD was 2090.9 +/- 369.1 cells/mm. There was no significant difference between the methods. CONCLUSIONS: White light confocal microscopy can be used for high-magnification imaging of corneas preserved in an eye bank with the 4 degrees C method, and images obtained permit evaluation of endothelium. Although the quality of confocal microscopy images of eye bank corneas is generally lower than that achieved with in vivo examinations, and although technical adaptations are needed for easier and safer application to corneas intended for transplantation, confocal microscopy is a promising new tool for evaluation of collected corneas.     

In vivo evaluation of corneal structure changes after refractive procedures

PURPOSE: The aim of the study was to evaluate and compare in vivo the corneal structure changes after refractive procedures (PRK, LASIK, LASEK). MATERIAL AND METHOD: The analysed group of patients consisted of 226 corneas in 126 patients, who underwent correction of myopia and myopic astigmatism, using the procedures: PRK (120 eyes), LASIK (56 eyes) and LASEK (50 eyes). The photoablation of the corneas was performed with the Excimer Laser MEL 60 and MEL 70 G-Scan Aesculap Meditec. Postoperative observations were made using a confocal microscopes Confoscan P4 (Tomey) and ConoScan 2 (Fortune Technologies). The evaluations were performed in the early (up to 3 months) and late postoperative period (after PRK and LASIK-up to 2 years; after LASEK-up to 6 months). RESULTS: The confocal microscopy revealed some changes within the corneal epithelium and anterior part of stroma after PRK, LASIK and LASEK. After PRK, there was increased desquamation of superficial epithelial cells in early postoperative period. These cells were elongated after LASIK and LASEK procedures. After PRK and LASEK, the Bowman's membrane was absent in the central part of the cornea, during the whole observation period. After all these procedures, the anterior part of the corneal stroma in the ablation zone, showed increased background illumination of collagen fibres and an irregular pattern of elongated keratocytic nuclei, in the early postoperative period. No scar tissue--"haze" was found in cases after LASEK, what may occur after PRK procedure. The findings kept changing in the course of the follow up time. CONCLUSIONS: Confocal microscopy enables in vivo monitoring of changes, which occur in the corneal structure after refractive procedures; this facilitates the evaluation of corneal healing. LASEK is the least invasive refractive procedure, which allows prompt stabilization of the corneal structure.