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Results for “Confocal microscopy”

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At least 109 records · Page 6Linked to original sources
PubMed2001

Evaluation of corneal stromal changes in vivo after laser in situ keratomileusis with confocal microscopy.

PURPOSE: To assess by in vivo confocal microscopy the modifications of the corneal stroma after laser in situ keratomileusis (LASIK) for myopia. DESIGN: Nonrandomized comparative (self-controlled) trial. PARTICIPANTS: Sixteen eyes of 13 patients were examined before surgery and at days 8, 30, and 90, and 9 eyes were examined at 6 months postoperatively using an in vivo confocal microscope. TESTING/INTERVENTION: Stromal morphologic changes, keratocyte density, flap thickness, and subclinical haze were evaluated and compared at different time points. LASIK was performed with a Flapmaker microkeratome (Solan Ophthalmic products, Jacksonville, FL) and a Lasersight LSX excimer laser (LaserSight Technologies Inc., Winter Park, FL). MAIN OUTCOME MEASURE: Confocal microscopy results. RESULTS: Microfolds at the Bowman's layer were found in most eyes, as well as variable reflectivity particles (pa) located at the interface level in all eyes examined postoperatively. The density of these particles significantly decreased with time with, respectively, 504 +/- 101 pa/mm2 at day 8 and 380 +/- 111 pa/mm2 at day 30 (P = 0.003), 332 +/- 100 pa/mm2 at month 3 and 312 +/- 40 pa/mm2 at month 6. The mean flap and the activated-cells area thicknesses were, respectively, 102 +/- 26 microm and 61 +/- 19 microm and showed significant negative correlation (P < 0.0001). The intensity of the added peak (47.3 microm 8.6%), corresponding to the subclinical haze, realized by Z-scan measure, was also negatively correlated with flap thickness (P = 0.01). Keratocyte (k) density quantified in the posterior stroma significantly increased from day 0 (480 +/- 67 k/mm2) to day 8 (701 +/- 41 k/mm2, P < 0.0001 compared with day 0) and day 30 (917 +/- 143 k/mm2, P = 0.0006, compared with day 0) but significantly decreased at 3 months postoperatively (597 +/- 56 k/mm2, P < 0.0001 compared with day 30) to reach the initial level at month 6 (502 +/- 41 k/mm2, nonsignificant compared with day 0). There was no correlation between preoperative or postoperative spherical equivalent and the density of particles, keratocytes, and the haze intensity. CONCLUSIONS: This study confirms the presence of microfolds and particles at the interface level, as well as subclinical impairment. Evaluation of keratocyte density constitutes a major contribution of confocal microscopy toward an understanding of the keratocyte response to corneal wound healing after corneal refractive surgery. Moreover, flap thickness seems to be involved in the postoperative cellular activation with a higher response when thin.

Adult↗
PubMed2005

Three-dimensional analysis of complex branching vessels in confocal microscopy images.

The characteristic of confocal microscopy (CM) vascular data is that it contains many tiny vessels with branching and complex structure. In this work, an automated method for quantitative analysis and reconstruction of cerebral vessels from CM images is presented in which the extracted centerline of the vessels plays the key role. To assess the efficiency and accuracy of different centerline extraction methods, a comparison among three fully automated approaches is given. The centerline extraction methods studied in this work are a snake model, a path planning approach, and a distance transform-based method. To evaluate the accuracy of the quantitative parameters of vessels such as length and diameter, we apply the method to synthetic data. These results indicate that the snake model and the path planning method are more accurate in extracting the quantitative parameters. The efficiency of the approach in clinical applications is then confirmed by applying the method to real CM images. All three methods investigated in this work are accurate enough to correctly distinguish between normal and stroke brain data, while the snake model is the fastest for clinical applications. In addition, three-dimensional visualization, reconstruction, and characterization of CM vascular images of rat brains are presented.

Brain↗
PubMed2004

Adaptive image-processing technique and effective visualization of confocal microscopy images.

A common observation about confocal microscopy images is that lower image stacks have lower voxel intensities and are usually blurred in comparison with the upper ones. The key reasons are light absorption and scattering by the objects and particles in the volume through which light passes. This report proposes a new technique to reduce such noise impacts in terms of an adaptive intensity compensation and structural sharpening algorithm. With these image-processing procedures, effective 3D rendering techniques can be applied to faithfully visualize confocal microscopy data.

Algorithms↗
PubMed1991

The application of scanning confocal microscopy in cartilage research.

Scanning confocal microscopy has been used in conjunction with immunofluorescent localization to address two areas of debate in cartilage research. With the enhanced resolution and optical sectioning capability of this new technique, we have demonstrated that type IX collagen is preferentially located in an area around the chondrocyte, even in young cartilage. We have also shown that cathepsin B production is not confined to de-differentiated chondrocytes. The advantages and versatility of scanning confocal microscopy have thus clearly been demonstrated.

Animals↗
PubMed1999

Automated quantification of keratocyte density by using confocal microscopy in vivo.

PURPOSE: To compare keratocyte density determined by using confocal microscopy with keratocyte density determined in the same corneas by histology. METHODS: Digital en face images of central corneas were recorded three times by using confocal microscopy in vivo in six New Zealand White rabbits. Bright objects (keratocyte nuclei) in the images were automatically identified by using a custom algorithm to estimate total and regional stromal keratocyte densities. The corneas were then excised, fixed, and sectioned in a sagittal plane for histology. Keratocyte nuclei were manually counted from digitized images of 50 histologic sections per cornea. Total and regional keratocyte densities were estimated from the histologic sections by using stereologic methods based on nuclei per unit area, mean nuclear diameter, and section thickness. Histologic cell densities were corrected for tissue shrinkage. RESULTS: By confocal microscopy, total keratocyte density was 39,000 +/- 1,200 cells/mm3 (mean +/- SE; n = 6); cell density was 47,100 +/- 1,300 cells/mm3 in the anterior stroma and decreased to 27,900 +/- 2,700 cells/mm3 in the posterior stroma (P = 0.004). Analysis of the three separate confocal images of each cornea produced repeatable total cell densities (mean coefficient of variation = 0.035). By histology, total keratocyte density was 37,800 +/- 1,100 cells/mm3, not significantly different from that estimated by confocal microscopy (P = 0.43); anterior cell density was 48,300 +/- 900 cells/mm3 and decreased to 29,400 +/- 900 cells/mm3 posteriorly (P < 0.001). CONCLUSIONS: Rabbit keratocyte density estimated by automated analysis of confocal microscopy images in vivo is repeatable and agrees with keratocyte density estimated from histologic sections.

Algorithms↗
PubMed1999

Confocal microscopy in the iridocorneal endothelial syndrome.

AIMS: To report the appearances of iridocorneal endothelial (ICE) syndrome from real time, white light confocal microscopy. METHODS: Three consecutive patients, each with ICE syndrome, were examined prospectively. Corneal specular and confocal microscopic examinations were performed in all three patients. In the first patient, a penetrating keratoplasty was performed and the cornea was examined by light and scanning electron microscopy. No surgery was performed in the remaining two patients. RESULTS: In the first patient corneal oedema prevented endothelial specular microscopy. Confocal microscopy performed before penetrating keratoplasty successfully revealed abnormal epithelial-like endothelial cells. Histological examinations of the cornea following penetrating keratoplasty revealed the presence of multilayered endothelial cells with epithelial features (microvilli). In the remaining two patients, specular microscopy showed the presence of ICE cells with typical dark/light reversal. Confocal microscopy demonstrated groups of endothelial cells with epitheloid appearances. In all three patients, the contralateral endothelial appearance was normal by specular and confocal microscopy, except for moderate endothelial polymegathism in one patient. Epithelial-like endothelial cells were characterised by prominent nuclei on confocal microscopy. CONCLUSIONS: The application of confocal microscopy indicates that the ICE syndrome is characterised by epitheloid changes in the endothelium. Confocal microscopy may be used to diagnose the ICE syndrome by demonstrating epithelial-like endothelial cells with hyperreflective nuclei. This technique is especially of value in cases of corneal oedema, since specular microscopy may fail to image the endothelium in such cases.

Aged↗
PubMed2001

Confocal microscopy and microbial viability detection for food research.

Confocal microscopy offers several advantages over other conventional microscopic techniques as a tool for studying the interaction of bacteria with food and the role of food microstructure in product quality and safety. When using confocal microscopy, samples can be observed without extensive preparation processes, which allows for the evaluation of food without introducing artifacts. In addition, observations can be made in three dimensions without physically sectioning the specimen. The confocal microscope can be used to follow changes over a period of time, such as the development of the food structure or changes in microbial population during a process. Microbial attachment to and detachment from food and food contact surfaces with complex three-dimensional (3-D) structures can be observed in situ. The fate of microbial populations in food system depends on processing, distribution, and storage conditions as well as decontamination procedures that are applied to inactivate and remove them. The ability to determine the physiological status of microorganisms without disrupting their physical relationship with a food system can be useful for determining the means by which microorganisms survive decontamination treatments. Conventional culturing techniques can detect viable cells; however, these techniques lack the ability to locate viable cells in respect to the microscopic structures of food. Various microscopic methods take advantage of physiological changes in bacterial cells that are associated with the viability to assess the physiologic status of individual cells while retaining the ability to locate the cell within a food tissue system. This paper reviews the application of confocal microscopy in food research and direct observation of viable bacteria with emphasis on their use in food microbiology.

Bacteria↗
PubMed1998

Confocal microscopy of cystic disorders of the corneal epithelium.

OBJECTIVE: This study aimed to describe the morphology of cystic disorders of the corneal epithelium by confocal microscopy. DESIGN: The study design was a prospective evaluation of confocal microscopic images of patients with cystic corneal disorders. PARTICIPANTS: Thirteen patients (19 eyes) were included. The corneal disorders included four patients with corneal decompensation (Fuchs' dystrophy), five patients with epithelial basement membrane dystrophy (e.g., Cogan's microcystic and map-dot dystrophies), one patient with Meesmann's dystrophy, and three patients with recurrent erosion syndrome of unknown etiology. Confocal images of diseased corneas were compared with those of ten normal control eyes (ten subjects). INTERVENTION: All patients were examined by slit-lamp biomicroscopic analysis and confocal microscopic analysis (Tomey, Erlangen-Temmenlohe, Germany). Image analysis was used to identify the corneal epithelial structures correlated with the corresponding pathology. MAIN OUTCOMES MEASURES: Confocal microscopy was used to assess the size, shape, light scatter, and reflection of the cysts. RESULTS: Slit-lamp examination results showed corneal epithelial cystic lesions in all cases. Confocal microscopy was able to identify cystic lesions in 9 (69.2%) of 13 patients. Of the four patients in whom lesions could not be found by confocal microscopy, three had recurrent erosion syndrome and the other one had epithelial basement membrane dystrophy. The confocal images were compatible with the clinical and histologic pictures of the disease. Normal control eyes did not show any epithelial lesion, either by biomicroscopy or confocal microscopy. CONCLUSIONS: Confocal microscopy provides an in vivo evaluation of cystic epithelial corneal lesions. This study shows that confocal microscopy is suitable for examining cystic lesions of the corneal epithelium. Nevertheless, it is not as sensitive as biomicroscopy in detecting cystic lesions in certain corneal conditions.

Corneal Diseases↗
PubMed2004

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry.

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.

Animals↗
PubMed1997

Evaluation of infectious crystalline keratitis with confocal microscopy in a case series.

We sought to determine whether there are unique findings in infections crystalline keratitis (ICK) examined by confocal microscopy and if confocal microscopy is predictive for bacteriology in ICK. A retrospective review of consecutive patients with a presumed diagnosis of ICK by slit-lamp examination was performed. These patients were then examined with confocal microscope and cultured. Sixteen patients were identified by biomicroscopy. Average age was 71 years; 12 of 16 patients were women; 10 of 16 had prior penetrating keratoplasty; and 12 of 16 were taking topical steroids. Confocal microscopy revealed a variable appearance to the crystals in the corneal stroma. Eight of 16 patients had distinct needle-like deposits at varying depths in the stroma, and eight had amorphous deposits grouped at different levels of the stroma. The results of confocal microscopic examination resembled the reported histopathology with clusters of deposits, but its current resolution does not allow identification of bacterial morphology. There was no correlation of morphology with culture results. Organisms were recovered in 12 of 16 patients by culture. In 10 of 16 patients, the infection was successfully treated with topical antibiotics, usually cefazolin. Crystal morphology of ICK can be observed by confocal microscopy. No pathognomonic, single pattern for this disease is seen with the confocal microscope. The latter may be an aid in determining the clinical response to treatment.

Adult↗
PubMed2006

In vivo confocal microscopy of pigmented conjunctival tumors.

PURPOSE: To analyze the appearance of conjunctival pigmented tumors as seen by in vivo confocal microscopy. METHODS: Twenty-eight pigmented conjunctival tumors including 6 nevi, 13 acquired melanoses, 7 conjunctival melanomas, and 2 extrascleral growths of uveal melanomas were examined by in vivo confocal microscopy using the Heidelberg Retina Tomograph (HRTII)/Rostock Cornea Modul (RCM). Confocal images were analyzed using predefined criteria by an observer masked to final histological diagnosis and a preliminary diagnosis was established. After excision, histology and immunohistochemistry using antibodies against S-100, Melan-A, HMB-45, Ki-67, CD3, and CD68 were performed in all specimens and compared with in vivo confocal images of the same lesions. RESULTS: Confocal microscopy images confirmed typical histopathological features of conjunctival pigmented tumors. Nest or diffuse collections of medium-sized uniform hyper- or hyperreflective cells in the stroma and stromal cysts lined with a multilayered epithelium were visible in 100% of conjunctival nevi. Small dendritic cells were typically observed in 100% of primary acquired melanoses (PAM) without atypia and in 2 out of 6 nevi. Large networks of hyperreflective dendritic cells were present in 100% of PAM with atypia. Whereas images of PAM without atypia and secondary complexion-associated melanosis showed hyperreflective granules confined to the basal epithelium in 67% of lesions, PAM with atypia presented with hyperreflective granules and patches throughout the epithelium in all cases. Malignant melanomas of the conjunctiva and extrascleral growths of uveal melanomas demonstrated large hyperreflective cells with prominent nuclei and nucleoli. In vivo confocal microscopy showed a sensitivity of 89% and a specificity of 100% to establish the correct diagnosis of conjunctival melanoma compared with histology. CONCLUSIONS: High correlations were found between in vivo confocal microscopy using near-infrared laser light and histology in the diagnosis of pigmented conjunctival lesions. In vivo confocal microscopy seems to be a valuable new tool in the differential diagnosis and follow-up of pigmented conjunctival tumors. It does not replace histology, but may assist in performing guided biopsy in tumors suspected clinically and/or with in vivo microscopy. In addition, in vivo confocal microscopy may support the clinical diagnosis of extrascleral involvement in uveal melanoma.

Adolescent↗
PubMed1994

Guidance of filopodial extension by fibronectin-rich extracellular matrix fibrils during avian gastrulation. A study using confocal microscopy.

We have used double-label confocal microscopy to examine the relationships between the orientation of filopodial extension in mesoderm cells and the orientation of fibronectin-rich extracellular matrix fibrils during chick embryo gastrulation. We fluorescently labeled mesoderm tissue dissected from donor embryos by immersion in carboxyfluorescein and then grafted it into unlabeled host embryos at the same stage of gastrulation. After further incubation, the host embryos were fixed, the endoderm removed, and the extracellular matrix was immunostained with antibodies to fibronectin conjugated to Texas Red. We found that both the general shape of the mesoderm cells and the orientation of filopodial extension were influenced by the surrounding matrix fibrils. Elongated shape was associated with individual fibrils which impinge on only one side of the cell. Similarly, filopodial extension followed a single fibronectin-rich fibril, although filopodia were also observed to be channeled between pairs of parallel fibrils. Cells attached to non-aligned regions of substratum showed no polarity. The mesoderm cells themselves apparently synthesize their own fibronectin, and deposit this on the cell surface not attached to the substratum. We conclude that individual fibronectin-rich substratum fibrils, in the size range 0.7-2.8 microns, are able to exert contact guidance on the mesoderm cells, despite the production of endogenous fibronectin by the cells themselves. These results support the contention that contact guidance is a physiological mechanism influencing the orientation and directionality of cells during the morphogenetic movements of embryogenesis.

Animals↗
PubMed2004

[Morphologic characteristics of cornea in Fuchs endothelial dystrophy observed by confocal microscopy].

OBJECTIVE: To analyze the image of corneas with Fuchs endothelial dystrophy using confocal microscopy in vivo. METHODS: Central corneas of 38 eyes (19 patients aged 33 approximately 76 years) were examined using scanning slit confocal microscopy in vivo after being diagnosed with Fuchs endothelial dystrophy. They were divided into two groups namely symptomatic (19 eyes) and asymptomatic (19 eyes). The control group comprised of 30 normal eyes. The images were analyzed visually for morphology of corneas and the densities of cells, as well as the diameter of the guttae and the corneal nerves were counted by NAVIS software. RESULTS: (1) In the symptomatic groups, confocal microscopy revealed guttae in 19 eyes. The diameter of the guttae ranged from 20 to 60 micro m. The density of endothelial cells was statistically significant compared with the control group (t = 18.74, P < 0.01). Descemet's membrane was thickened in 9 eyes. Dark bands 6.0 approximately 10.8 micro m in diameter were detected in 14 eyes. All eyes showed a diffuse increased light reflection in the stroma.17 eyes exhibited an abnormal Bowman's layer: multiple focal bright reflection. 10 eyes revealed normal corneal nerves. The differences between posterior and anterior keratocyte densities in the control and study groups were not statistically significantly different (t = 0.854, 1.173; P = 0.38, 0.24 respectively). (2) In the asymptomatic groups, less number of guttae could be seen in the endothelial layer. The diameter of the guttae was 15 approximately 40 micro m. The densities of endothelial cells were not significantly different compared with control group (t = 1.998, P = 0.053). Other layers of the cornea exhibited no other abnormal phenomenon. The densities of endothelial cells between symptomatic and asymptomatic groups were statistically significantly different (t = 8.352, P < 0.01). CONCLUSIONS: Morphologic characteristics of cornea are observed by confocal microscopy in Chinese patients with Fuchs endothelial dystrophy. Preliminary results demonstrate that there is no significant difference between Chinese and western patients. Confocal microscopy allows to diagnose Fuchs dystrophy and visualize endothelial cells within the swollen cornea. And it is especially useful for patients whose endothelial cells can not be seen with non-contact specular microscopy.

Adult↗
PubMed2005

Use of confocal microscopy in comparative studies of vertebrate morphology.

Laser scanning confocal microscopy provides a means to acquire and analyze images of complex morphological structures and to help place molecules or cells of interest in their proper morphological context. Confocal microscopy is a form of fluorescence microscopy that sharpens the images collected by visualizing the light from only one plane of focus. This allows for the collection of multiple focal planes in what is called a z-stack, which provides three-dimensional data. Five steps that any investigator using a confocal microscope should follow are described: (1) labeling and (2) mounting of specimens for viewing, (3) optimizing the image on the confocal, and (4) collecting and (5) analyzing of confocal image data. We describe three specific protocols incorporating these steps from our work on vertebrate inner ear development. The first two describe a collection of z-stacks in living, fluorescently labeled, and intact embryos. The second protocol is for time-lapse imaging of multiple focal planes at each time point. The third protocol describes confocal imaging of preserved material double labeled with antibodies and by retrograde labeling of neurons via axonal uptake. Finally, three alternative or complementary approaches to standard confocal microscopy are described and discussed.

Anatomy, Comparative↗
PubMed1995

Confocal microscopy of cells implanted into tissue blocks: cell migration in long-term histocultures.

In three-dimensional tissues in vivo, cells find themselves in a unique, heterogeneous microenvironment among various cellular and noncellular elements. Cells are greatly affected by and contribute to their physical and chemical microenvironments. However, live cells are currently studied predominantly in homogeneous monolayer cultures where newly established contacts might be fundamentally different from contacts in vivo. Several systems have been suggested to simulate the three-dimensional environment of real tissue. In this report, we describe a new system for studying cell behavior inside real tissues in vitro. By fluorescently labeling mouse tumor cells, them implanting them into cultured tissue blocks (histocultures), we have observed cellular location and followed their locomotion, within tissues in vitro for days. We discuss the potential of the described system for studying different aspects of cell behavior in a nativelike microenvironment.

Animals↗