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PubMedDate not supplied

Confocal microscopy of the living eye.

Confocal microscopy is an imaging paradigm that allows optical sectioning of almost any material with increased axial and lateral spatial resolution and better image contrast. We have applied this technology to the study of the living eye of cats, albino rabbits, and humans. The technique allows in vivo, noninvasive, real time images of the eye at magnifications (630x) which allow resolution of anatomical detail at the cellular level. In this paper we report details of our current instrument techniques and some of our results. The past development, present state-of-the-art, and projected future advances and applications of this novel microscopy are discussed. Preliminary observations are reported for all layers of the cornea, the limbus, and wound-healing responses in single animals.

Analog-Digital Conversion↗
PubMed2005

Clinical and microstructural analysis of patients with hyper-reflective corneal endothelial nuclei imaged by in vivo confocal microscopy.

The purpose of this study was to determine the significance of hyper-reflective corneal endothelial nuclei imaged by in vivo confocal microscopy. A retrospective analysis was performed using a database of 505 patients that had undergone in vivo confocal microscopy of the cornea. All subjects with hyper-reflective endothelial nuclei were identified and these images were analysed to determine corneal endothelial cell density and morphology. The clinical notes of these patients were reviewed and corresponding data regarding corneal thickness was obtained from a related database of Orbscan II pachymetry. Hyper-reflective endothelial nuclei were identified in 41 eyes of 39 (7.7%) patients. Diagnoses included previous cataract surgery or penetrating keratoplasty, posterior polymorphous dystrophy, Fuchs' endothelial dystrophy and irido-corneal endothelial syndrome. No patients with clinically normal corneas exhibited bright endothelial nuclei. The mean endothelial cell density in this group was 1325+/-872 cells mm(-2) and endothelial density was below age-adjusted normal values in 69.2% of patients. Both cellular polymegathism (coefficient of variation of cell area 33.9+/-7.4%) and cellular pleomorphism were noted (51.8+/-9.0% hexagonal cells). The mean central corneal thickness was 582+/-52 microm. There was no significant difference in endothelial density and morphology compared to cases that had low endothelial density but did not exhibit bright nuclei. In conclusion, this study is the first to investigate the significance of bright endothelial nuclei detected by in vivo confocal microscopy. The strong association with corneal disease states suggests that the most likely explanation for this appearance is the alteration in cellular/nuclear morphology, composition or function.

Cataract Extraction↗
PubMed2004

Mechanical properties of polyelectrolyte-filled multilayer microcapsules studied by atomic force and confocal microscopy.

By using a combination of atomic force and confocal microscopy, we explore the deformation properties of multilayer microcapsules filled with a solution of strong polyelectrolyte. Encapsulation of polyelectrolyte was performed by regulation of the multilayer shell permeability in water-acetone solutions. The "filled"capsules prepared by this method were found to be stiffer than "hollow" ones, which reflects the contribution of the excess osmotic pressure to the capsule stiffness. The force-deformation curves contain three distinct regimes of reversible, partially reversible, and irreversible deformations depending on the degree of compression. The analysis of the shape of compressed capsules and of the inner polyelectrolyte spacial distribution allowed one to relate the deformation regimes to the permeability of the multilayer shells for water and inner polyelectrolyte at different stage of compression.

Acetone↗
PubMed1997

Expression of the five somatostatin receptor (SSTR1-5) subtypes in rat pituitary somatotrophes: quantitative analysis by double-layer immunofluorescence confocal microscopy.

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 +/- 9.7% of GH cells followed by SSTR2 in 42 +/- 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 +/- 4.7% and 18 +/- 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5 +/- 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

Animals↗
PubMed2006

Virtual biopsy of the joint tissues using near-infrared, reflectance confocal microscopy. A pilot study.

Standard noninvasive imaging techniques applied to joints provide gross morphological features, insufficient for assessing histological detail. On the other hand, biopsying is invasive, time consuming, and may involve unwanted processing artifacts. Near-infrared reflectance confocal microscopy is a technique that allows serial, high-resolution optical sectioning through intact tissues without employing exogenous fluorescent stains. The aim of this work was to evaluate the potential utility of near-infrared reflectance confocal microscopy for providing immediate histological information on meniscus, articular cartilage, epiphyseal plate, bone, muscle, and tendon. Images from near-infrared reflectance confocal microscopy were compared with mirror routine histology sections. Characteristic architectural features were readily visualized in the three dimensions of space. Additionally, the use of experimental contrast agents highlighted the localization of nuclei. Limitations include penetration depth and minor optical artifacts. In conclusion, near-infrared reflectance confocal microscopy is a useful technique for immediate, nondestructive, serial "virtual" sectioning through intact tissues, being thus a potential adjunct to current imaging techniques in orthopedics.

Animals↗
PubMed2002

Keratocyte density in keratoconus. A confocal microscopy study(a).

PURPOSE: To estimate keratocyte density in human corneas with keratoconus by confocal microscopy. DESIGN: Prospective, observational cohort study. METHODS: Twenty-nine unscarred corneas of 19 patients with keratoconus and 29 corneas of 19 controls matched for age (+/-3 years) and contact lens wear were examined by using confocal microscopy. Images were recorded from the full-thickness central cornea. A masked observer manually counted bright objects (keratocyte nuclei) in images without motion blur. Cell densities in anteroposterior stromal layers of keratoconus corneas were compared with densities in corresponding layers of control corneas. RESULTS: In keratoconus patients, age 40 +/- 15 years (mean +/- standard deviation), keratocyte density was 19% lower in those who wore contact lenses (16,894 +/- 4032 cell/mm(3), n = 12) than in those who did not wear contact lenses (20,827 +/- 4934 cell/mm(3), n = 17, P =.03). In control patients, age 39 +/- 16 years, there was no difference in keratocyte density between those who wore contact lenses (n = 12) and those who did not wear contact lenses (n = 17, P =.80). Among contact lens wearers, keratocyte density was 25% lower in keratoconus corneas (16,894 +/- 4, 032 cell/mm(3), n = 12 [9 = rigid gas-permeable lenses, 3 = soft lenses]) than in control corneas (22,579 +/- 2, 387 cell/mm(3), n = 12 [3 = rigid gas-permeable lenses, 9 = soft lenses], P =.002), the result of cell density being lower in the most anterior keratocyte layer (P =.001) and the layers between 0% to 10% (P <.001), 67% to 90% (P <.001), and 91% to 100% (P <.001) of stromal thickness. Among noncontact lens wearers, there was no difference in cell density between keratoconus and controls (P =.41). CONCLUSION: Keratocyte density is decreased in the anterior and posterior stroma of keratoconus patients who wear contact lenses.

Adult↗
PubMed2003

In vivo confocal microscopy of Fleck dystrophy and pre-Descemet's membrane corneal dystrophy.

PURPOSE: To assess the value of in vivo confocal microscopy (CM) in the diagnosis of Fleck dystrophy and pre-Descemet's membrane corneal dystrophy. METHODS: Case report of two patients. Standard slit-lamp and ophthalmic examination and in vivo CM were performed on both patients. The thickness of the cornea and the morphology of the corneal epithelium, stroma, endothelium, and subbasal nerves were evaluated by confocal microscopy. RESULTS: Biomicroscopy revealed bilateral, fine, dust-, and flour-like opacities in the corneal stroma for the Fleck dystrophy patient. In the pre-Descemet's membrane corneal dystrophy patient, biomicroscopy showed opacities larger than those in the first patient. Both patients were then examined by in vivo CM. Confocal microscopy of the Fleck dystrophy showed intracellular deposits throughout the stroma. In pre-Descemet's membrane corneal dystrophy, however, these and the extracellular deposits were observed immediately anterior to Descemet's membrane. The thicknesses of the corneas were 560 and 650 microm for Fleck and pre-Descemet's membrane corneal dystrophy, respectively. The surface epithelium, subbasal nerves, and endothelium showed normal morphology in both patients. CONCLUSION: In vivo CM is a valuable tool in diagnosing rare corneal dystrophies when the final diagnosis is difficult to obtain with conventional methods.

Adult↗
PubMed2002

Cornea in Marfan disease: Orbscan and in vivo confocal microscopy analysis.

PURPOSE: To investigate corneal thickness, curvature, and morphology with the Orbscan Topography System I (Bausch & Lomb, Inc., Salt Lake City, UT) in patients with Marfan syndrome (MFS) and to study MFS with in vivo confocal microscopy. METHODS: This prospective, clinical, comparative case series included 60 eyes of 31 patients with MFS and 32 eyes of 17 control subjects. First, biomicroscopic examination was conducted to search for ectopia lentis. Then, mean keratometry and ocular refractive power were calculated by the autokeratorefractometer. In each group, the Orbscan System I mean (and mean simulated) keratometry and pachymetric measurements (at the central location and at eight midperipheral locations) were obtained and compared, and correlations were established. In vivo confocal microscopy was performed to evaluate tissue morphology and Z-scan analysis of 14 thin MFS corneas compared with 14 control corneas. RESULTS: A significant decrease (ANOVA, P < 0.0001) of mean simulated keratometry measurement appeared in the MFS group (sim K, 40.8 +/- 1.4 D) compared with the control group (42.9 +/- 1.1 D). Pachymetry in the MFS group was significantly decreased (P < 0.0001) compared with that in the control group, in the center (respectively, 502 +/- 41.9 microm and 552 +/- 23.6 microm) and the eight midperipheral locations. Ectopia lentis was highly linked with mean keratometry and pachymetry (P < 0.0001). Confocal microscopy performed on MFS-affected thin corneas confirmed the corneal thinning and showed an opaque stromal matrix, and Z-scan profiles were abnormal with increased stromal back scattering of light. CONCLUSIONS: MFS is known to be associated with a flattened cornea. This study demonstrated an association with corneal thinning and described confocal microscopy findings in this syndrome.

Adolescent↗
PubMed1996

Measurement of enamel demineralization using microradiography and confocal microscopy. A correlation study.

Substantial amounts of tooth minerals are lost during dental caries formation. Transversal microradiography, a well-accepted method used to quantify mineral loss, is a time-consuming technique which requires a thin enamel section (100 microns) and involves the use of x-rays. In an attempt to solve these difficulties, a procedure has been developed in which a human tooth specimen with demineralized enamel is cut in half (HT), stained with a fluorescent dye (rhodamine B) and analyzed using a laser scanning confocal microscope. A series of three studies was conducted to correlate measurements of enamel demineralization obtained from enamel thin (100 microns) sections (TS) using transversal microradiography with three parameters (area of the lesion; total and average dye fluorescence intensities) measured on the same TS or on a thicker section (HT) of the same specimen by laser scanning confocal microscopy. Results showed that a 0.1 mM rhodamine B solution provided the most adequate imaging conditions for confocal microscopy. Pearson's correlation coefficients, calculated between microradiography and confocal microscopy data obtained using a 0.1 mM rhodamine B solution, were: delta Z vs. HT lesion area = 0.95; delta Z vs. HT total fluorescence = 0.80; delta Z vs. HT average fluorescence = 0.74; delta Z vs. TS lesion area = 0.95; delta Z vs. TS total fluorescence = 0.74; delta Z vs. TS average fluorescence = 0.55. All these correlations coefficients were statistically significant (p < 0.01). It is concluded that in enamel demineralization studies statistically significant correlations exist between parameters measured using transversal microradiography and parameters quantified using confocal microscopy.

Analysis of Variance↗
PubMed1996

Quantitative imaging in electron and confocal microscopies for applications in biology.

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be complemented by non-parametric approaches (e.g., different variants of multivariate statistical techniques). The other kind of series consists of multiple mappings of a specimen. We describe several new tools for the study and quantification of the co-location, with potential application to multiple mappings in microanalysis or in fluorescence microscopy.

Image Processing, Computer-Assisted↗
PubMed2005

In vivo confocal microscopy in a patient with keratopigmentation (corneal tattooing).

PURPOSE: To report in vivo corneal confocal microscopic findings in a patient with keratopigmentation (corneal tattooing). PATIENTS AND METHODS: A 64-year-old woman who received keratopigmentation for bilateral corneal opacity 53 years ago was examined by confocal microscope. RESULTS: By confocal microscopy, scattered highly reflective particles in a geographic pattern were observed in the superficial stroma near Bowman layer. In addition, clusters of highly reflective granules were in the mid- to superficial stroma. CONCLUSIONS: In vivo confocal microscopy can be used for monitoring keratopigmentation. The findings may serve to distinguish keratopigmentations from other pigmented corneal lesions.

Carbon↗
PubMed2002

In vivo confocal microscopy in primary congenital glaucoma with megalocornea.

PURPOSE: To report the microscopic findings of congenital glaucoma-related megalocornea using in vivo confocal microscopy. PATIENTS AND METHODS: Two consecutive adult patients presenting the typical features of glaucomatous megalocornea underwent a complete ophthalmologic examination. The first patient presented with progressive glaucoma with bilateral megalocornea. The second patient's left eye was affected by megalocornea without actual evidence of glaucoma, whereas the right eye was healthy. Both patients were examined using a new-generation scanning slit corneal confocal microscope. RESULTS: In both patients, confocal microscopy revealed a mild reduction of keratocyte density in the mid and rear stroma, a particular abnormal "clew-shaped" morphology of stromal nerves, and the presence of discontinuous hyperreflective structures overhanging the endothelial layer at the level of the Descemet membrane. The endothelium showed severe polymegethism, pleomorphism, and a markedly decreased cell density, and focal cellular lesions were noted. CONCLUSION: Confocal microscopy is a diagnostic tool used to evaluate microscopic aspects of Haab striae and endothelial morphologic changes in glaucomatous megalocornea. Unsuspected alterations, such as nerves abnormalities and focal endothelial tractions by scar tissue, were observed.

Adult↗
PubMed2005

NMR and confocal microscopy studies of the mechanisms of burst drug release from PLGA microspheres.

Pulsed-field gradient (PFG) NMR and confocal microscopy techniques have been used to study the structural evolution and drug release profile of poly(d,l-lactide-co-glycolide) (PLGA) microspheres over time during immersion in an aqueous phase. Variation of the drying process used in the synthesis of the PLGA microspheres has been found to significantly influence the degree of permeability of the spheres to water. PFG NMR has been used to study the change in the cavity sizes within the pore structure of the microspheres over time following initial immersion. In these studies, the temperature of the secondary emulsion, used in the sphere synthesis, has been found to significantly change the temporal evolution of the pore structure. Confocal microscopy studies of the release of a model drug from within the microspheres suggest that the rate-limiting step in drug release is the swelling rate of the polymer matrix, and that the mechanism may be a percolation process. These studies also showed that the local rate of drug release is heterogeneously distributed across a microsphere, and thus, strictly, cannot be modelled as purely a simple diffusive release process from a sphere.

Algorithms↗
PubMed2006

Confocal microscopy of subepithelial infiltrates occurring after epidemic keratoconjunctivitis.

PURPOSE: To describe the confocal microscopic findings in subepithelial infiltrates associated with epidemic keratoconjunctivitis (EKC). METHODS: Observational case report. RESULTS: A 14-year-old Saudi girl with keratoconus developed subepithelial infiltrates (SEIs) after the onset of bilateral EKC. Confocal microscopy of the left cornea 8 weeks after the onset of EKC showed many highly reflective dendritic cells at the level of basal epithelium, epithelial basement membrane zone, and anterior stroma, as well as many highly reflective fusiform and round cells within the anterior stroma, with deceasing density in progressively deeper layers of the stroma. These findings were not present on confocal microscopy that had been performed 2 weeks before the onset of EKC. CONCLUSION: Confocal microscopic examination of SEIs after EKC provides evidence of an inflammatory response localized to the basal epithelium and anterior stroma of the central cornea.

Adolescent↗
PubMed2006

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus.

PURPOSE: To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. METHODS: Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. RESULTS: Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. CONCLUSIONS: This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.

Adult↗
PubMed2006

Confocal microscopy of subepithelial infiltrates occurring after epidemic keratoconjunctivitis.

PURPOSE: To describe the confocal microscopic findings in subepithelial infiltrates (SEIs) associated with epidemic keratoconjunctivitis (EKC). METHODS: Observational case report. RESULTS: A 14-year-old Saudi girl with keratoconus developed SEIs after the onset of bilateral EKC. Confocal microscopy of the left cornea 8 weeks after the onset of EKC showed many highly reflective dendritic cells at the level of basal epithelium, epithelial basement membrane zone, and anterior stroma, as well as many highly reflective fusiform and round cells within the anterior stroma, with decreasing density in progressively deeper layers of the stroma. These findings were not present on confocal microscopy that had been performed 2 weeks before the onset of EKC. CONCLUSION: Confocal microscopic examination of SEIs after EKC provides evidence of an inflammatory response localized to the basal epithelium and anterior stroma of the central cornea.

Adolescent↗
PubMed2007

In vivo laser confocal microscopy findings for Bowman's layer dystrophies (Thiel-Behnke and Reis-Bücklers corneal dystrophies).

OBJECTIVE: To investigate microstructures in patients with genetically confirmed Bowman's layer dystrophies (Thiel-Behnke or Reis-Bücklers corneal dystrophy) using an in vivo laser scanning confocal microscope. DESIGN: Single-center, prospective, comparative small case series. PARTICIPANTS: Two patients from one pedigree (a 29-year-old woman and 58-year-old man) with Thiel-Behnke corneal dystrophy (Arg555Gln [R555Q] heterozygous missense mutation of human transforming growth factor beta-induced [TGFBI] gene) and 3 patients from one pedigree (a 70-year-old woman, 58-year-old man, and 14-year old man) with Reis-Bücklers corneal dystrophy (Arg124Leu [R124L] heterozygous missense mutation of the TGFBI gene) were examined. Two patients with Reis-Bücklers corneal dystrophy exhibited recurrence after corneal transplantation. TESTING: All patients were examined by slit-lamp biomicroscopy. The center and the peripheral cornea of both eyes also were examined by in vivo laser scanning confocal microscopey. Image analysis was used to identify the corneal epithelial and stromal deposits correlated with each disorder. MAIN OUTCOME MEASURES: Selected images of the corneal layers were evaluated qualitatively for the shape and degree of light reflection of the deposits. RESULTS: In each dystrophy, distinct characteristic deposits were observed in the epithelium and Bowman's layer, respectively, by in vivo laser scanning confocal microscopy. In Thiel-Behnke corneal dystrophy, the deposits in the epithelial basal cell layer showed homogeneous reflectivity with round edges accompanying dark shadows. In contrast, deposits in Reis-Bücklers corneal dystrophy in the same cell layer showed extremely high reflectivity from small granular materials without any shadows in all cases. In each dystrophy, Bowman's layer was replaced totally with pathological materials; the reflectivity of those materials is much higher in Reis-Bücklers corneal dystrophy than in Thiel-Behnke corneal dystrophy. CONCLUSIONS: In vivo laser scanning confocal microscopy is capable of identifying in vivo corneal microstructural changes related to Thiel-Behnke and Reis-Bückler corneal dystrophy with a higher resolution than is available with slit-lamp biomicroscopy or in vivo white-light confocal microscopy. As a result, this device may enable differentiation of Thiel-Behnke and Reis-Bücklers corneal dystrophy in vivo. In vivo laser scanning confocal microscopy also may be a valuable tool for further research into the corneal dystrophies, especially to follow the natural course.

Adolescent↗
PubMed2005

Confocal microscopy: imaging cervical precancerous lesions.

OBJECTIVES: We explore the clinical potential of reflectance and fluorescence confocal microscopy to image the morphologic and biochemical changes associated with precancer, in order to aid in the detection and diagnosis of cervical dysplasia. METHODS: Cervical epithelial tissue samples imaged ex vivo or in vivo were obtained from M. D. Anderson Cancer Center and Lyndon B. Johnson Hospital in Houston, Texas. Confocal reflectance microscopy was used to image ex vivo cervical biopsies and in vivo cervical tissue. Confocal fluorescence microscopy was used to image ex vivo cervical tissue slices. RESULTS: We present reflectance and fluorescence confocal images of cervical tissue demonstrating the ability to differentiate between normal and abnormal cervical tissue. CONCLUSIONS: We believe that there is significant clinical potential for confocal microscopy to provide a sensitive and specific method for cervical precancer detection.

Biopsy↗