W. Matthew Petroll

Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease.

BACKGROUND The purpose of this article is to introduce the practicing ophthalmologist to the optical principles and images produced by a tandem scanning confocal microscope (recently approved by the Food and Drug Administration for general clinical use). The tandem scanning confocal microscope allows real-time viewing of structures in the living cornea at the cellular level in four dimensions (x, y, z, and time). METHODS Nine patients (2 males, 7 females), ranging in age from 7 to 52 years, were examined. Images were recorded on super VHS videotape, digitized and processed on a computer workstation, and photographed for presentation. RESULTS Two-dimensional (x, y) 400 x 400-microns images (9-microns z-axis thickness) are presented for normal corneal structures and for the clinical conditions of herpetic keratitis, wound healing after myopic excimer ablation, Acanthamoeba infection, corneal dystrophies (granular, Reis-Buckler), contact lens abrasion, and the irido-corneal endothelial syndrome. CONCLUSION Clinical confocal microscopy has the unique potential of providing noninvasive assessment of corneal injury and disease at the cellular level that is not available currently from other technologies.

Cell Motility and Mechanics in Three-Dimensional Collagen Matrices

Fibrous connective tissues provide mechanical support and frameworks for other tissues of the body and play an integral role in normal tissue physiology and pathology. Three-dimensional collagen matrices exhibit mechanical and structural features that resemble fibrous connective tissue and have become an important model system to study cell behavior in a tissue-like environment. This review focuses on motile and mechanical interactions between cells—especially fibroblasts—and collagen matrices. We describe several matrix contraction models, the interactions between fibroblasts and collagen fibrils at global and subcellular levels, unique features of mechanical feedback between cells and the matrix, and the impact of the cell-matrix tension state on cell morphology and mechanical behavior. We develop a conceptual framework to explain the balance between cell migration and collagen translocation including the concept of promigratory and procontractile growth factor environments. Finally, we review the significance of these concepts for the physiology of wound repair.

Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy

PURPOSE The authors establish, for the first time, observer-independent quantification of stromal thinning, epithelial thickness, and corneal haze after excimer laser photorefractive keratectomy (PRK) using a unique, new form of in vivo confocal microscopy. METHODS Rapid, continuous z-scans of high-resolution confocal images, termed confocal microscopy through focusing (CMTF), were performed in the central corneal area of 17 patients before and 1 month after PRK for low- to moderate-grade myopia (-2.88-9.13 diopters [D]). Corneal, epithelial, and stromal thickness measurements and an objective haze estimate were obtained from each CMTF scan by digital image analysis. RESULTS Epithelial thickness averaged 51 +/- 4 microns before and 45 +/- 10 microns 1 month post-PRK (P < 0.005), whereas stromal thinning ranged from 20 to 154 microns, representing a direct estimate of the actual photoablation depth. Corneal thickness averaged 560 +/- 36 microns before PRK and 462 +/- 52 microns at 1 month. The change in corneal thickness correlated closely with the change in spherical equivalent refraction (r = 0.94, P < 0.0001); linear regression analysis revealed a value of 14.3 microns corneal thinning per diopter of correction. A significant correlation was found between the objective CMTF haze estimate and a clinical haze grading obtained by slit-lamp examination (r = 0.73, P < 0.001). CONCLUSIONS Confocal microscopy through focusing is a new, powerful in vivo tool that enables quantitative, unbiased evaluation of PRK procedures over time by providing epithelial and stromal thickness analysis, photoablation depth assessment, and unbiased haze measurement. The method is uniquely valuable in the pre- and postoperative assessment of PRK patients and for determining the optimal treatment strategy, especially in assessing refractive and visual outcomes in individual cases.

Effects of rigid and soft contact lens daily wear on corneal epithelium, tear lactate dehydrogenase, and bacterial binding to exfoliated epithelial cells☆

OBJECTIVE To determine the effects of lens type and oxygen transmissibility on human corneal epithelium during daily lens wear (DW). DESIGN Prospective, randomized, double-masked, single-center, parallel treatment groups clinical trial. PARTICIPANTS Two hundred forty-six patients fitted with: (1) high oxygen-transmissible soft lenses (n = 36), (2) hyper oxygen-transmissible soft lenses (n = 135), and (3) hyper oxygen-transmissible rigid gas-permeable (RGP) lenses (n = 75). INTERVENTION Irrigation chamber to collect exfoliated epithelial surface cells, confocal microscopy, and tear collection at baseline, 2 weeks, and 4 weeks of DW. MAIN OUTCOME MEASURES (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal epithelial surface cells, (2) central epithelial thickness, (3) superficial epithelial cell area, (4) epithelial surface cell exfoliation, and (5) tear lactate dehydrogenase (LDH). RESULTS Four weeks of DW with the high oxygen-transmissible soft lens significantly increased PA binding from baseline 6.55 +/- 3.01 to 8.75 +/- 3.05 bacteria per epithelial cell (P < 0.01). By contrast, hyper oxygen-transmissible soft lens wear increased binding significantly less (6.13 +/- 2.45 to 7.62 +/- 3.06; P < 0.01), whereas hyper oxygen-transmissible RGP lens wear demonstrated no significant changes (5.91 +/- 2.40 to 6.13 +/- 2.17; P = 0.533). No significant change in central epithelial thickness was found after 4 weeks of DW in either soft lens; however, the epithelial thickness decreased by 9.8% (P < 0.001) with RGP lens wear. Epithelial cell surface area increased 3.3% and 4.1% with the high and hyper oxygen-transmissible soft lenses, respectively, and 10.5% with the hyper oxygen-transmissible RGP lens (P < 0.001). Epithelial desquamation significantly decreased in all groups (P < 0.001). Tear LDH levels increased for all test lenses (P < 0.001). CONCLUSIONS Increased PA binding induced by wear of a conventional soft lens material is significantly greater than that induced by the new hyper oxygen-transmissible soft silicone hydrogel lens during DW. However, both soft materials showed significant increases in PA binding as compared with baseline controls. By contrast, hyper oxygen-transmissible RGP lens DW did not increase PA binding significantly. Taken together, these findings suggest for the first time both an oxygen effect as well as a difference between soft and rigid lens types on PA binding in DW.

Adaptive effects of 30-night wear of hyper-O2 transmissible contact lenses on bacterial binding and corneal epithelium: A 1-year clinical trial☆

OBJECTIVE To determine effects of lens type and oxygen transmissibility on human corneal epithelium during extended wear (EW). DESIGN Prospective, randomized, double-masked, single-center, parallel treatment groups, 1-year clinical trial. PARTICIPANTS One hundred seventy-eight patients completed the study: (1) high-O(2) soft lens (6-night [N] EW) (n = 27); (2) hyper-O(2) soft lens (6N-EW, n = 33) or (30N-EW, n = 66); and (3) hyper-O(2) rigid gas-permeable lens (RGP) (30N-EW, n = 52). INTERVENTION Irrigation chamber to collect exfoliated corneal surface cells, confocal microscopy, and tear collection at baseline, 1, 3, 6, 9, 12 months of EW. MAIN OUTCOME MEASURES (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness; (3) superficial epithelial cell area; (4) epithelial surface cell exfoliation; and (5) tear lactate dehydrogenase. RESULTS Quantitative evidence demonstrated increased binding of PA to human exfoliated corneal epithelial cells during the first 3 months of soft lens EW; the control high-O(2) test lens showed significantly higher bacterial binding (P < 0.05). Binding activity gradually decreased thereafter and returned to baseline after 9 and 12 months. The corneal epithelium demonstrated enlargement of surface cell size, thinning of central epithelium, and a significant decrease in surface cell shedding (P < 0.05). Remarkably, there was subsequent partial adaptive recovery in cell shedding and epithelial thickness but not surface cell size. There was no significant difference between 6N and 30N continuous wear of the hyper-O(2) soft lens for all outcome measures. Importantly, hyper-O(2) RGP lens wear did not show significantly increased PA binding during 1 year. CONCLUSIONS This study establishes three important new findings: (1) hyper-O(2) soft lens EW produces significantly less PA binding than the lower O(2) soft lens with no significant difference in PA binding with 6N versus 30N EW of the hyper-O(2) soft lens; (2) there is a remarkable adaptive recovery after 6 months with all soft lens wear with gradual return to prelens PA binding levels and partial recovery of other outcome measures for all test lenses EW except surface cell size; (3) 30N EW of the hyper-O(2) RGP lens produced no significant increases in PA binding over 1 year. Taken together, these results suggest that introduction of new hyper-O(2) transmissible lens materials into clinical use may offer safer EW, and future epidemiologic studies of ulcerative infectious keratitis should consider both lens type and time in lens EW in any incidence/risk analysis.

The Relation between Contact Lens Oxygen Transmissibility and Binding of Pseudomonas aeruginosa to the Cornea after Overnight Wear

PURPOSE To assess adverse effects of contact lens-induced hypoxia on the rabbit cornea in vivo and determine the relation between binding of Pseudomonas aeruginosa and oxygen transmissibility for rigid and hydrogel lenses. METHODS Six rigid lenses with Dk/Ltotal values between 0 and 97 x 10(-9) (cm/second) (ml O2/ml mmHg) and four hydrogel lenses (Dk/Ltotal 9, 20, 39, 51) were tested. All lenses had 14.0-mm diameters and a thickness (parallel) of 0.12 or 0.15 mm. Tear lactate dehydrogenase activity and tandem scanning confocal microscopy determinations were performed after the lens was worn for 24 hours. Binding of P. aeruginosa then was separately determined by the colony-forming unit method. Scanning electron microscopy was used to confirm in vivo tandem scanning confocal microscopy findings. RESULTS Lens oxygen transmissibility determines binding of P. aeruginosa to the cornea after the lens is worn for 24 hours; epithelial damage produced by lenses of lower Dk/Ltotal appears to be the dominant biologic factor for P. aeruginosa binding and not lens rigidity. CONCLUSIONS These results suggest that the risk of P. aeruginosa keratitis developing with overnight wear will be enhanced significantly for contact lenses with Dk/Ltotal values less than 50 x 10(-9) (cm/second) (ml O2/ml mmHg) (human equivalent oxygen percentage < or = 15%), and this risk will increase with further decreases in oxygen transmissibility. Because no hydrogel lenses approved by the Food and Drug Administration are available with oxygen transmission at this level, patients should be made aware of the increased risk of infectious keratitis associated with the overnight wear of current extended wear hydrogel lenses. Results of this study also demonstrate that quantitative clinical tandem scanning confocal microscopy imaging and tear lactate dehydrogenase activity measurements can provide prospective, noninvasive methods for assessing the ongoing interaction between contact lens and cornea in vivo.

Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

PURPOSE: To investigate the feasibility of using femtosecond‐pulse lasers to produce second‐harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two‐photon confocal microscopy using a variable‐wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second‐harmonic‐generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second‐harmonic‐generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowmans layer, suggesting an anchoring function. CONCLUSIONS: Using two‐photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.

In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy

OBJECTIVE To characterize morphologic changes in corneas of patients with recurrent erosion syndrome or epithelial basement membrane dystrophy using in vivo confocal microscopy. DESIGN Observational case series PARTICIPANTS Fourteen eyes of eight patients with diagnosed epithelial basement membrane dystrophy and 13 eyes of seven patients with recurrent erosion syndrome were examined. METHODS Slit-lamp examination and in vivo confocal microscopy. The pathologic findings are presented as digitized images obtained from video tape recorded during the confocal microscopy. MAIN OUTCOME MEASURES The morphology of corneal surface epithelial cells, basal epithelial cells, subbasal nerve plexus, Bowmans layer, stromal keratocytes, and endothelium was analyzed. RESULTS The surface epithelium was intact in all but two eyes. One cornea (a basement membrane disorder with clinically visible dots) had multinucleate surface epithelial cells, and one eye with recurrent corneal erosions showed a freely floating surface epithelium sheet in the tear fluid. Patients in both groups showed islets of highly reflective cells with presumed intracellular deposits surrounded by normal cells in the basal epithelial cell layer. The basal epithelial cell area also showed other pathologic changes, including drop-shaped configurations, streaks, or ridges. Folding of the Bowmans layer was also observed in both groups. Anterior keratocytes showed signs of activation (highly reflective nuclei with visible processes) in some of the patients regardless of the clinical diagnosis, and in recurrent erosions even increased deposition of abnormal extracellular matrix in the anterior stroma was suspected. Posterior corneal keratocytes and endothelium appeared normal when examined. The subbasal nerve plexus showed various pathologic changes, such as short or strangely shaped nerve fiber bundles, decreased numbers of long nerve fiber bundles, only faintly visible long nerve fiber bundles (instead of the normally observed long parallel running interconnected bundles), or increased amounts of Langerhans cells, but only one patient (with recurrent erosion syndrome) lacked the subbasal nerve plexus. CONCLUSIONS In vivo confocal microscopy of corneas with recurrent erosions or epithelial basement membrane dystrophy showed deposits in basal epithelial cells, subbasal microfolds and streaks, damaged subbasal nerves, or altered morphology of the anterior stroma. Confocal microscopy cannot replace biomicroscopy in making a specific diagnosis, but it sometimes helps the diagnosis in corneas that appear normal under a biomicroscope.

Effects of daily and overnight wear of a novel hyper oxygen-transmissible soft contact lens on bacterial binding and corneal epithelium: A 13-month clinical trial

OBJECTIVE To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear. DESIGN Prospective, randomized, double-masked, single-center, 13-month clinical trial. PARTICIPANTS One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98). One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n = 55, 30 nights). TESTING Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW. MAIN OUTCOME MEASURES (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH). RESULTS Daily wear with control lens increased PA binding from 5.90 +/- 2.60 to 7.81 +/- 3.04 bacteria per cell (P < 0.01); HOTL wear increased PA binding significantly less (5.31 +/- 1.87-5.98 +/- 2.26; P < 0.01). Daily wear produced no significant changes in CET or SCA. Significant decreases in DESQ were seen with both lenses with no significant intergroup differences. Tear LDH increased significantly in DW with HOTL wear versus control (P = 0.0017), but not after 1 month of subsequent EW (P = 0.533). One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear (P < 0.01); binding adaptively decreased thereafter, returning to baseline at 9 to 12 months. Lens EW produced significantly enlarged SCA, thinning of CET (except 6-night HOTL wear), and decreased DESQ (P < 0.01). Some adaptive recovery was seen with CET and DESQ, but not SCA; importantly, the data indicated no significant difference between 6- or 30-night EW for all outcomes. CONCLUSIONS Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs. 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year. These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.

Specular Microscopy, Confocal Microscopy, and Ultrasound Biomicroscopy Diagnostic Tools of the Past Quarter Century

Purpose. To identify, characterize, and illustrate the most important past and future potential contributions of specular, confocal, and ultrasound biomicroscopy to clinical diagnosis and research applications in the cornea from the past 25 years. Methods. Specular microscopy, in vivo tandem scanning confocal microscopy (TSCM), scanning slit confocal microscopy (SSCM), and high-frequency ultrasound biomicroscopy are examined. Results and Conclusions. This review demonstrates the abilities and limitations of three powerful new in vivo imaging modalities to resolve the cellular and structural layers of the cornea temporally and spatially in three or four dimensions, (x, y, z, t). Clinical pathological processes such as inflammation, infection, wound healing, toxicity, embryonic development, differentiation, and disease, which previously could be studied only under static ex vivo conditions, can now be dynamically evaluated over time. Thus, with continued development and application in vivo, noninvasive microscopic techniques should provide exciting new insights into understanding the structure and function of not only the eye, but also other multicellular organ systems in health and disease. These new imaging paradigms are in the first rank of advances in medical science in the past quarter century.