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Results for “Confocal microscopy”

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PubMed2001

Microstructural assessment of rare corneal dystrophies using real-time in vivo confocal microscopy.

PURPOSE: To analyse and describe three cases of rare corneal dystrophy and highlight their in vivo microstructural features. METHODS: Subject 1 was diagnosed with a posterior stromal fleck corneal dystrophy Two of her three children were also affected. Subjects 2 and 3 exhibited an almost identical clinical appearance on biomicroscopic examination, such that both clinically were diagnosed as having pre-Descemet's dystrophies. All subjects underwent in vivo confocal microscopy and approximately 300 sequential digital images were obtained and analysed for each cornea. RESULTS: In vivo confocal microscopy of subject 1 demonstrated an abnormal appearance of numerous large ovoid particles, measuring 50-70 microm in diameter in the mid and posterior stroma as well as smaller hyperreflective dot-like intracellular deposits, of less than 1 microm diameter. Despite the near-identical clinical appearance, subjects 2 and 3 could be clearly differentiated by in vivo confocal microscopy. Subject 2 exhibited small, irregular, optically dense particles, mainly in the anterior stroma, whereas subject 3 possessed classical involvement of the stroma immediately adjacent to Descemet's membrane, with numerous regular small, hyperreflective particles. CONCLUSIONS: The ability of in vivo confocal microscopy to localize and accurately measure various elements in different corneal layers may help to resolve whether abnormalities are intra- or extracellular, and aid clearer differentiation of rare corneal disorders.

Adult↗
PubMed1999

Confocal microscopy of dentinal tubules in human tooth stained with alizarin red.

The present study was designed to analyze the structures of dentinal tubules by confocal microscopy. Undecalcified ground sections of human teeth were stained with alizarin red in 0.1% KOH aqueous solution, and examined by confocal microscopy. Alizarin red stained dentinal tubules, interglobular dentine, granular layer of Tomes, and the surface of dentine. Interglobular dentine was seen between the outer and middle layers of coronal dentine. At the outer layer of coronal dentine, the dentinal tubules were thin and showed numerous branches. At the middle layer of coronal dentine, dentinal tubules displayed two types. The type I tubules are the dentinal tubules that do not show any nodular structures and the type II tubules are the dentinal tubules that appear bamboo-like with many nodules. In the cross section through the type II tubules, the nodules appeared as fine circular tubules surrounding the dentinal tubules. The circular tubules of nodules adhered to one side of the dentinal tubules. When the fluorescence images were compared with the images taken by transmission light mode, the fluorescence of dentinal tubules was seen at the inner surface of dentinal tubules, and the fluorescence of nodules was seen at interface between peritubular and intertubular dentine. Most of the dentinal tubules were of the type II tubules in the teeth from older individuals, whereas the type II tubules were scarce in the teeth from younger individuals. At the inner layer of coronal dentine, the dentinal tubules have no nodules and branches were scarce. The dentinal tubules of radicular dentine were different from those of coronal dentine. Most of the dentinal tubules were the type I tubules. Numerous fine branches were seen at the outer and middle layers of radicular dentine. No interglobular dentine was seen in the root except at the cervical part, and the granular layer of Tomes was also positive with alizarin red. At the cervical part of the root, interglobular dentine was present and the dentinal tubules displayed types I and II.

Adult↗
PubMed1997

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.

Adult↗
PubMed2004

Chromatic confocal microscopy with a finite pinhole size.

Chromatic confocal microscopy has the advantage of short measurement times because of its parallel depth scan. As most white-light sources have limited optical output power, light-efficient setups are necessary. Using an extended detection pinhole is one way to improve light efficiency. We have calculated the effect of extended pinholes in chromatic confocal setups. We found that, for certain pinhole sizes, the FWHM of the confocal signal is nearly constant over a large wavelength interval.

Microscopy, Confocal↗
PubMed2003

Organotypic slices in vitro: repeated, same-cell, high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live, transfected cerebellar neurons by confocal microscopy.

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports. This system permits cultures to be repeatedly observed both by phase contrast microscopy and, upon biolistic transfection, by laser confocal microscopy using 40x, 60x, and 100x water-immersion objectives, at high resolution. Upon co-transfection with two plasmids, differentiation of the same transfected Purkinje neurons was followed across in vitro development for periods of up to 10 days. Despite the relative thickness of the slice culture, even small, punctate, nuclear signals, were detectable. The results show that Purkinje neurons in cerebellar slices explanted from postnatal day 2 mice, developed cytotypically, although some were ectopically located. In contrast, Purkinje neurons in slices from postnatal day 6 cerebella developed in an organotypic manner. It is concluded that this culture system serves as an ideal tool for applications in experimental biology where high resolution tracking of cellular signals, over extended time periods, is of interest.

Animals↗
PubMedDate not supplied

Three-dimensional confocal microscopy of the human cornea in vivo.

This paper reviews the literature from 1990 to 2000 and evaluates the seminal investigations performed with the confocal microscope on the in vivo human cornea. Our pedagogical technique is to illustrate both the advantages and the problems associated with occular confocal microscopy by way of annotated examples. Confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level. The ability to provide high-resolution, real-time images of the full thickness of the cornea gives the clinician and the researcher an important new tool for the investigation of the cornea.

Cell Count↗
PubMed2002

Using tandem scanning confocal microscopy to predict the status of donor kidneys.

Tandem scanning confocal microscopy (TSCM) is a noninvasive form of vital microscopy that can be used to evaluate superficial uriniferous tubules in living kidneys. Because TSCM has a number of advantages over conventional microscopic examination of renal biopsies, the present study was undertaken to determine whether the histopathological images obtained by TSCM correlate with post-transplant renal function. The kidneys of New Zealand male rabbits were harvested, flushed with Euro-Collins solution, and stored at 0-2 degrees C for periods of 24, 48, 67 and 72 h prior to transplantation. TSCM observation of the kidneys prior to their transplantation revealed characteristic histopathological changes of the superficial proximal convoluted tubules that correlated closely with subsequent post-transplant renal function. These observations indicate that TSCM may be of significant value in evaluating the status of donor kidneys prior to their transplantation.

Animals↗
PubMed2003

Confocal microscopy in ocular chrysiasis.

PURPOSE: To describe the presence of gold salt deposits in the corneal tissue of a patient treated with gold sodium thiomalate through confocal microscopy and to relate our findings to temporary corneal dynamics. METHODS: A patient who had been treated with gold sodium thiomalate for 6 years was referred from the Rheumatology Unit of our center for an ophthalmologic examination. At the time of examination she had been treatment-free for 9 years. Besides visual acuity, the examination included slit-lamp biomicroscopy, intraocular pressure, and fundoscopy. Confocal microscopy performed using a Confoscan P4 instrument with a 40x lens to obtain 15 images per second on an S-VHS video system (Panasonic) for subsequent analysis. RESULTS: The basic layered structure of the cornea was unaffected, but the presence of highly reflective particles was noted. These particles were interpreted as gold deposits in the corneal stroma, especially in the anterior and middle stroma. CONCLUSION: Confocal microscopy is a useful method of evaluating the deposition of gold salts in tissues, particularly the cornea. Its future use will help gain a deeper understanding of corneal metabolism.

Antirheumatic Agents↗
PubMed2006

Dendritiform cells found in central cornea by in-vivo confocal microscopy in a patient with mixed bacterial keratitis.

PURPOSE: To report the in-vivo confocal microscopic findings of dendritiform cells in the central corneal epithelial layer in a case of mixed bacterial keratitis associated with severe immunologic reaction. DESIGN: Observational case report. METHODS: A 25-year-old male suffered from contact lens-related mixed bacterial keratitis with a dense eccentric immune ring. In-vivo confocal microscopy was performed to study the different layers of the central and peripheral cornea in the lesion and the fellow eye. RESULTS: Several dendritiform cells were found in the basal and superficial epithelial layers of the central cornea in the lesion eye, which was also the area of the dense immune ring formation. No such cells could be identified in the fellow eye or the unaffected area of the lesion eye. CONCLUSION: Corneal dendritiform cells, possibly dendritic or Langerhans cells, can be identified in severe corneal immunologic conditions using in-vivo confocal microscopy. The role of these cells in ocular immunity is interesting and needs further clarification.

Adult↗
PubMed2001

[Application of confocal microscopy in the study of the relationships between astrocytes and blood vessels in the retina].

OBJECTIVE: To study the rabbit retina astrocytes by using immunofluorescence techniques together with the confocal microscopy. MATERIAL AND METHODS: The rabbit retinas were processed with an anti-glial fibrillary acidic protein (anti-GFAP) for astrocyte detection and with propidium lobide for nuclear staining. RESULTS: Confocal microscopy allows for three-dimensional reconstruction of astroglial cells, the performance of double staining procedures with superposition of images corresponding to each chromogen, the exchange of observation axes for each slide and finally the performance of serial optic sections that indicate the exact cell location and their relationship with adjacent structures, eliminating the background signals. CONCLUSIONS: The confocal microscopy provides detailed information about tridimensional morphology and the location of the astrocytes in the rabbit retina. The astrocytes associated with the nerve fiber bundles are located in the nerve fiber layer. The type III perivascular astrocytes are located between the intravitreous capillaries close to the internal limiting membrane of the retina. The type I perivascular astrocytes are found in the retinal face of the intravitreal capillaries, these being the neurons which are the most distant from the retina.

Animals↗
PubMed1998

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy.

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Endocytosis↗
PubMedDate not supplied

Vital confocal microscopy in bone.

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.

Adipocytes↗
PubMed2005

[In vivo confocal microscopy observation and evaluation of sensation in diabetic corneas].

OBJECTIVE: To test the changes of corneal sensation in diabetic patients and to observe the diabetic corneas using confocal microscopy. METHODS: One hundred and thirty-two diabetic patients were enrolled in this study. The diabetic history and medication were recorded. The FBG and HbA(1c) were checked for every patient. The patient's eye was systemically checked, including the IOP, visual acuity, cornea sensation, lenses, and the fundus. Two hundred patients of other diseases were enrolled as controls. The cornea of 85 patients and 56 controls were checked by the confocal microscopy. The right eyes of both patients and controls were used for analysis. RESULTS: The cornea sensation of diabetic patients was significantly depressed as compared to that of the controls (P < 0.01). The depression of cornea sensation was related to the course of the disease, the presence of diabetic retinopathy and the degree of the diabetic retinopathy (P < 0.05). The confocal microscopy revealed that there was a higher corneal epithelium edema rate (11.76%) in diabetic patients than that of the controls (1.79%) (P < 0.05). The nerve fiber density of the diabetic patients was decreased and there were some morphological changes in the nerve fiber of diabetic patients, such as sparse, slim, less branches, less long fibers, edema and zigzag of the fibers. CONCLUSION: The depression of the corneal sensation in diabetic patients is probably caused by the reduction and the degeneration of the corneal nerve fibers.

Adolescent↗
PubMed2006

[Confocal microscopy in corneal dystrophies: a comparison between confocal slit scanning (ConfoScan P2) and laser scanning microscopy (Rostock Cornea Modul-HRT II)].

BACKGROUND: The object of this work was a comparison of confocal images of corneal dystrophies made by a slit scanning microscope versus a laser scanning microscope. MATERIAL AND METHODS: Using the Rostock Cornea Modul-HRT II as a confocal laser scanning microscope the images of five patients with some epithelial, stromal and endothelial corneal dystrophies were acquired. The pictures were compared qualitatively with those taken by the slit scanning microscope "ConfoScan P2" from corresponding pathologies. Also, the images of normal corneas of ten healthy persons were acquired for a qualitative comparison. RESULTS: Confocal images from both devices were able to provide significant helpful diagnostic findings about the corneal microstructure. Essential qualitative differences between the images of both devices used were not observed. Due to the additional hardware components and the software module for image acquisition, analysis and archiving, the RCM-HRT II is favoured over the "ConfoScan P2". Nevertheless, the evaluation in favour of the RCM-HRT II has to be confined because an optimised, user-friendly enhancement, the "ConfoScan 4" is currently available. CONCLUSION: Evaluating corneal dystrophies in vivo, an equivalent utility of both technical approaches has been observed.

Adult↗
PubMed2003

In vivo confocal microscopy of the human cornea.

AIMS: To describe the optics of in vivo confocal microscopy, its advantages over previous methods, and to summarise the literature that arose from its use for the observation of the human cornea. A critical review of the clinical usefulness of this new technology for the corneal examination is undertaken. METHODS: Confocal microscopes obtain increased resolution by limiting the illumination and observation systems to a single point. Rapid scanning is used to reconstruct a full field of view and allows for "real time" viewing. RESULTS: Coronal sections of the in situ epithelium, Bowman's membrane, stroma, and endothelium can be visualised at a resolution of 1-2 micro m. A backscattered light intensity curve allows objective measurements of sublayer thickness and corneal haze to be taken. In vivo confocal microscopy is therefore particularly useful in the areas of infective keratitis, corneal dystrophies, refractive surgery, and contact lens wear, where it aids in differential diagnosis and detection of subtle short and long term changes. Real time endothelial cell assessment can also be performed. CONCLUSION: Because of their ability to visualise living tissue at cellular levels, confocal microscopes have proved useful additions to the current clinical tools.

Cell Count↗
PubMed2004

Effects of benoxinate hydrochloride 0.4% on the morphological appearance of the cornea using confocal microscopy.

PURPOSE: To investigate whether benoxinate hydrochloride 0.4% used to make confocal microscopy more comfortable alters the morphology of the cornea as viewed with the confocal microscope. METHODS: Confocal microscopy was performed on both eyes of 10 subjects prior to instillation of either topical anaesthetic or non-preserved sterile saline, on two randomly ordered occasions. Images of all corneal layers were analysed quantitatively and qualitatively in a masked fashion. RESULTS: The images were similar in appearance in 5/10 subjects, there was greater clarity when anaesthetic was instilled in 4/10 subjects, and in the remaining subject there was greater clarity when saline was used. Anaesthetic had no influence on anterior keratocyte density (AKD), posterior keratocyte density (PKD) or endothelial cell density (ECD). CONCLUSIONS: Local anaesthetic does not affect corneal morphology as imaged using the confocal microscope. However, failure to use anaesthetic may lead to a degradation of image quality due to patient discomfort and excessive eye movements.

Journal Article↗
PubMed2000

Application of in vivo confocal microscopy to the objective evaluation of ocular irritation induced by surfactants.

An ocular irritation test using confocal laser scanning ophthalmoscopy has been developed in which corneal lesions subsequent to instillation of surfactants are specifically marked by fluorescein and assessed by digital image processing. The sum of the observed fluorescent corneal areas is taken into account as an endpoint of ocular irritation. Eight currently used nonionic, cationic and anionic surfactants were applied onto the cornea of rabbits and mice, four times per day during 3 days at various concentrations. Benzalkonium chloride, a cationic surfactant, at a concentration range of 0.01-0.5%, was tested in the same manner. The cornea was evaluated in vivo for ocular tolerance by confocal microscopy. In both rabbits and mice, the test revealed following irritation rankings: cationic>anionic>nonionic surfactants. Furthermore, in both animal models, the ocular damage increased with the concentration of benzalkonium. The test was sensitive enough to detect ocular microlesions at concentrations of surfactants as low as 0.01% for benzalkonium. These findings demonstrate the usefulness of confocal microscopy for the non-invasive, in situ evaluation of ocular tolerance.

Animals↗
PubMed2002

Confocal microscopy in Meretoja syndrome.

PURPOSE: To study the correlation between severity of clinical systemic symptoms and the degree of stromal haze and visibility of epithelial and stromal nerves on scanning slit confocal microscopy examination in Meretoja syndrome. METHODS: Three patients with Meretoja syndrome were examined by slit-lamp microscopy and scanning slit confocal microscopy. RESULTS: Increased symptoms and abnormal slit-lamp findings correlated positively with confocal microscopic stromal haze intensity and inversely with visibility of epithelial and stromal nerves on confocal examination. A prominent deposit, presumably amyloid, was observed contiguous to a stromal nerve of an affected cornea. In a severely affected cornea, no stromal or epithelial nerves were seen. CONCLUSION: The results of this study suggest that Meretoja syndrome causes corneal nerve damage and eventual degeneration that correlates with the degree of clinical involvement.

Adolescent↗