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PubMed2006

Confocal microscopy for Nocardia keratitis.

PURPOSE: To assess the role of confocal microscopy for in vivo detection of Nocardia in patients with microbial keratitis. DESIGN: Retrospective interventional case series. PARTICIPANTS: Three patients with microbiologically proven Nocardia keratitis. INTERVENTION: Confocal microscopy. METHODS: We performed confocal microscopy in microbiologically proven cases of Nocardia asteroides keratitis. A masked observer examined the images. For better understanding of the images, we also performed confocal microscopy on a blood agar culture plate with Nocardia growth. RESULTS: Confocal microscopy of infiltrated cornea revealed highly reflective, short, thin branching filaments with bright inflammatory cells in the background. The filaments were seen clearly at the edge of the infiltrate. In scans with faint images, these filaments became more visible on inversion of bright and dark components. Confocal microscopy of cultured organisms also revealed filamentous beaded structures with a morphology identical to that of those seen in vivo. CONCLUSION: Nocardia, a filamentous bacterium, produces a distinct image on confocal microscopy. This in vivo examination technique may be useful in cases of deep-seated infiltrates where routine microbiology workup does not yield positive results.

Adult↗
PubMed1996

Confocal microscopy findings of Acanthamoeba keratitis.

PURPOSE: Tandem scanning confocal microscopy was performed on two patients with Acanthamoeba keratitis to provide images detailing characteristic findings of the disease. Although tandem scanning confocal microscopy of Acanthamoeba has been described in previous reports, Acanthamoeba keratitis has not been fully characterized with this instrument. In vivo confocal micrographs showed the double-walled structure of the Acanthamoeba cyst and associated radial keratoneuritis (perineuritis). METHODS: We reviewed the records of two patients with a clinical diagnosis of Acanthamoeba keratitis, one with culture-proven Acanthamoeba and the other with a suspected Acanthamoeba infection. Slit-lamp biomicroscopy and in vivo tandem scanning confocal microscopy were performed. The images obtained were compared with images from patients without corneal disease. RESULTS: High-contrast round bodies suggestive of Acanthamoeba cysts, as previously described, and irregular forms suggestive of Acanthamoeba trophozoites were found by tandem scanning confocal microscopy. Additionally, we showed conclusively that under certain circumstances (that is, corneal scarring) tandem scanning confocal microscopy can resolve the double-walled structure of the Acanthamoeba ectocyst surrounding the endocyst. Furthermore, radial keratoneuritis was demonstrated, consisting of an irregularly swollen nerve fiber with probable amoebic infiltration. CONCLUSIONS: Confocal microscopy can be a useful, noninvasive imaging technique helpful in the study, diagnosis, and treatment of Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1996

High-resolution confocal microscopy using synchrotron radiation.

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper.

Animals↗
PubMed2005

Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy.

Collagen structural organization plays an important role in the mechanical property of the vertebrate integument. Bufo ictericus and Rana catesbeiana integument was investigated by light microscopy and laser confocal microscopy. Collagenous elements of the dermis were statistical analyzed. The integument is formed by the keratinized squamous stratified epidermis supported by the dermis that is subdivided into the spongious layer with a loose arrangement, and the compact layer formed by collagenous fibers arranged compactly in a criss-crossed manner. Thick collagenous columns have a perpendicular trajectory, and are formed by the assembling of alternating collagenous lamellae in both animals. Short intercolumns of collagenous fibrils connecting collagenous lamellae obliquely or transversally are observed in R. catesbeiana dorsal integument. The present study provides evidences that B. ictericus and R. catesbeiana integument has well-organized compact dermis, constituted by collagenous lamellae in a plywood manner. The integument organization is in contrast to the literature in some aspects. This dermal arrangement is important to the biomechanical property of both anuran integuments.

Animals↗
PubMed2003

Stromal haze after laser in situ keratomileusis: clinical and confocal microscopy findings.

PURPOSE: To report clinical and confocal microscopy characteristics of haze-like opacities in corneas after laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany. METHODS: Eighteen eyes of 11 patients with clinically apparent corneal clouding were examined by slitlamp and confocal microscopy (Confoscan P4, Tomey) 1 to 9 months after primary LASIK or LASIK retreatment. RESULTS: Postoperative slitlamp examination showed faint, white, snowflake-like clouding at the interface level in all patients. One patient had folds and rather diffuse haze-like opacities. Confocal microscopy revealed highly reflective structures in the flap stroma and at the interface level in all patients, probably due to numerous activated keratocytes and their processes. The confocal microscopy appearance was similar to that of photorefractive keratectomy haze. CONCLUSION: Focal wound-healing reactions in the central flap stroma and interface resulting in significant keratocyte activation could be observed after LASIK.

Adult↗
PubMed1999

[Clinical diagnosis of fungal keratitis by confocal microscopy].

OBJECTIVE: To illustrate the value of confocal microscope in the diagnosis of fungal keratitis. METHODS: In vivo confocal microscopy was performed on 43 patients of suspected fungal keratitis. The results were compared to that of smears and biopsies. RESULTS: Fungal filaments were imaged in 31 of 43 patients' corneas. Fungal keratitis was diagnosed in 32 patients based on clinical signs and all the methods of examinations. The positive rate of confocal microscopy is 96.9%. CONCLUSION: Confocal microscopy can be a rapid, effective and noninvasive imaging technique, and it is helpful in the diagnosis, treatment and study of fungal keratitis.

Adolescent↗
PubMed1999

Assessing confocal microscopy systems for purchase.

Confocal laser scanning microscopy now has so many functions and applications that choosing a new multiuser confocal laser microscopy system can be a daunting task, particularly for a first-time buyer and new users of confocal microscopy. How does one determine which features are most appropriate for any particular laboratory, application, or imaging environment? Each confocal microscopy system has its own individual advantages and limitations, which ultimately defines its market niche. Here, we describe the features that differentiate the four confocal microscopy systems we assessed. The decisive factors in choosing a confocal microscopy system for our anatomical laboratory were user-friendly software for on-line acquisition and off-line processing; the working distances of objective lenses; applicability for a multiuser environment; interactions with company representatives; and turnaround times for questions, service, and accessories.

Computers↗
PubMed2005

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy.

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Biological Transport↗
PubMed1997

Assessment of the tear film with tandem scanning confocal microscopy.

PURPOSE: To evaluate the confocal microscopic appearance of the tear film by correlating the interference pattern created by the lipid surface with physiologic measurements of tear function and with clinic diagnosis. METHODS: A series of 53 patients was examined with a battery of tests to evaluate the tear film and with confocal microscopy to evaluate the interference pattern created by the lipid surface of the tear film. Assessment of the tear-film appearance involved five nonparametric scales: degree of debris in the tear film, variability of the interference pattern over time, linearity of the interference pattern, number of dry spots seen, and relative thickness of the lipid layer. For 10 other subjects, the confocal appearance of the tear film and evaporation from the ocular surface was tested for before and after meibomian gland expression. RESULTS: The confocal appearance correlated with multiple physiologic variables and clinical diagnosis. Patients with seborrheic meiboman gland dysfunction had a thicker lipid layer, greater variability, and more debris than did patients with obstructive meibomiam gland dysfunction. After meibomian gland expression, lipid thickness and linearity increased, whereas pattern variability and evaporation decreased significantly. The mean reduction in evaporation was 48%. CONCLUSIONS: Confocal microscopy can be used to examine the tear film, and its appearance correlates significantly with physiologic variables in normal-individuals and in disease states. The confocal appearance of the lipid interference patterns and evaporation from the ocular surface are altered after lipid expression from meibomian glands.

Adolescent↗
PubMed1997

Scanning slit confocal microscopy of fungal keratitis.

In vivo scanning slit confocal microscopy was performed in a patient with Fusarium solani keratitis. Morphologically distinctive abundant filamentous structures were observed intrastromally. Confocal microscopy of the culture plate growing F solani from the patient's corneal scraping revealed filaments morphologically similar to the filaments observed in vivo. After 1 week of medical therapy, subsequent confocal microscopy showed an increased load of filaments, supporting the decision to perform a penetrating keratoplasty. Confocal microscopy confirmed that all of the fungus was eradicated. This aided in the decision to administer corticosteroids and quickly discontinue antifungal agents.

Adolescent↗
PubMed2000

Optical aberrations and objective choice in multicolor confocal microscopy.

Refinements in design have simplified confocal microscopy to the extent that it has become a standard research tool in cell biology. However, as confocal microscopes have become more powerful, they have also become more demanding of their optical components. In fact, optical aberrations that cause subtle defects in image quality in wide-field microscopy can have devastating effects in confocal microscopy. Unfortunately, the exacting optical requirements of confocal microscopy are often hidden by the optical system that guarantees a sharp image, even when the microscope is performing poorly. Optics manufacturers provide a wide range of microscope objectives, each designed for specific applications. This report demonstrates how the trade-offs involved in objective design can affect confocal microscopy.

Animals↗
PubMed1999

Confocal microscopy in Bowman and stromal corneal dystrophies.

OBJECTIVE: To use confocal microscopy to demonstrate the similarity among three autosomal-dominant corneal dystrophies and the diversity of the deposit patterns within a single dystrophy. DESIGN: A prospective, comparative case series. PARTICIPANTS: Twenty patients (40 eyes) from 10 families suffering from Bowman or stromal dystrophy agreed to take part: 3 with Reis-Bückler dystrophy, 12 with granular dystrophy, and 5 with lattice type-I dystrophy. Of these, nine had recurrence in their grafts or after phototherapeutic keratectomy before the confocal examination. The confocal images of affected corneas were compared with those of ten normal control eyes (ten subjects). INTERVENTION: All patients were examined by slit-lamp biomicroscopy. Confocal microscopy was performed with Achroplan 40x/numeric aperture (NA) = 0.75 and 63x/NA = 0.9 water immersion objectives. Image analysis was used to identify the corneal epithelial and stromal deposits correlated with each disorder. MAIN OUTCOMES MEASURES: Selected images of the corneal layers were evaluated qualitatively for the size, shape, light scattering, and reflection of the deposits. RESULTS: Slit-lamp biomicroscopy showed stromal involvement in all affected eyes. Confocal microscopy identified epithelial deposits in 30% of the eyes and stromal deposits in all eyes. The deposits within the epithelium were revealed more clearly with the 63x/NA = 0.9 objective (higher numeric aperture). Some of the confocal findings near the Bowman layer were common for all three dystrophies. Normal control eyes showed no epithelial or stromal deposits, either by biomicroscopy or confocal microscopy. CONCLUSIONS: Confocal microscopy provides an in vivo evaluation of the deposits in the cornea, with a higher resolution than biomicroscopy. The confocal findings common to the three dystrophies may agree with previous hypotheses of the same genetic origin. It may be a useful adjunct to slit-lamp biomicroscopy, particularly when histopathologic studies cannot be performed.

Adolescent↗
PubMed2005

Ex vivo confocal microscopy of human LASIK corneas with histologic and ultrastructural correlation.

OBJECTIVE: To perform confocal microscopy on postmortem human LASIK corneas and correlate these findings to histologic and ultrastructure evaluations. DESIGN: Prospective, consecutive, observational case series. PARTICIPANTS: Ninety postmortem LASIK corneas (47 patients) were evaluated for histopathology, of which 22 consecutive corneas (12 patients) were also evaluated by confocal microscopy. Six normal corneas (3 patients) served as controls. METHODS: This observational case series involving 22 corneas from 12 patients with postoperative intervals from 1 month to 6.5 years after LASIK surgery were collected. The corneas were mounted in an artificial anterior chamber and perfused with balanced salt solution before confocal microscopy was performed on the center of the cornea. The corneas were then bisected and processed for light and transmission electron microscopy. RESULTS: Confocal microscopy, along with histologic and ultrastructural correlations, demonstrated that the most prevalent alterations in the centers of LASIK corneas were a slightly thickened epithelium caused by focal basal epithelial cell hypertrophic modifications, random undulations in Bowman's layer over the flap surface, and a variably thick hypocellular primitive stromal interface scar. By using confocal microscopy, the interface wound was easily identified in 100% of the cases because numerous brightly reflective interface particles were always present in the hypocellular primitive stromal scar. These particles were found primarily to consist of organic cellular constituents, some of which were transient in nature. CONCLUSION: After LASIK, active stromal wound healing in the central cornea results in the production of a hypocellular primitive stromal scar, whereas secondary tissue adjustments seem to cause the Bowman's layer undulations and the subsequent epithelial cell modifications. Most of the interface particles revealed by confocal microscopy in the region of the stromal scar are organic in nature and presumably innocuous to the cornea.

Adult↗
PubMed2006

Detection of UV-induced pigmentary and epidermal changes over time using in vivo reflectance confocal microscopy.

In vivo reflectance confocal microscopy (RCM) provides high-resolution optical sections of the skin in its native state, without needing to fix or section the tissue. Melanin provides an excellent contrast for RCM, giving a bright signal in the confocal images. The pigmented guinea-pig is a common animal model to study human pigment induction and modulation, as its tanning response is comparable to human tanning after exposure to ultraviolet radiation (UVR). We investigated the applicability of RCM to detecting UVR-induced pigmentary changes in this model. Animals were exposed to solar simulator radiation for 7 days. RCM was performed during the irradiation and follow-up period. Compared to non-irradiated skin, an increase in melanocyte size, dendricity, and number, as well as increased pigment in keratinocytes, was seen in the irradiated epidermis. Interestingly, these changes could be detected even before a tanning response was clinically visible. UVR-induced epidermal hyperplasia could also be detected and quantified. In conclusion, in vivo RCM is a sensitive non-invasive imaging technique that can repeatedly measure epidermal pigmentation and thickness, as demonstrated in the guinea-pig model. This technique should greatly enhance our appreciation of dynamic pigmentary changes in human or animal skin over time and in response to specific stimuli.

Animals↗
PubMed1998

In vivo tandem scanning confocal microscopy in acanthamoeba keratitis.

The in vivo confocal microscopy technique provides us with a real-time, non-invasive way of examining the human cornea. The most important advantage of this type of microscopy is to reveal the etiologic agents in infectious keratitis such as Acanthamoeba keratitis. We present several representative cases of Acanthamoeba keratitis, which were diagnosed in their early stages using in vivo confocal microscopy and managed based on that diagnosis. In our Acanthamoeba keratitis cases, highly-reflective round or ovoid organisms with a diameter of about 10-25 um were visualized distinctly against relatively-dark normal parenchymal structures, such as epithelial cells or keratocyte nuclei. Double-walled structures of Acanthamoeba cysts were clearly demonstrated in some cases. We can confirm that in vivo tandem scanning confocal microscopy is a powerful diagnostic tool for identifying the infecting organisms in Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1996

Fading correction for fluorescence quantitation in confocal microscopy.

Quantitative analysis in confocal microscopy meets with several problems such as fading of the fluorophore during scanning and attenuation of the fluorescence in thick tissue specimens. The present study reports a quantitative investigation of the enzyme uracil-DNA glycosylase (UDG), which removes uracils from DNA. For this study we developed a fading correction algorithm which takes into account both the number of prior scans in the specimen, and the differences in fading through the specimen from each prior scan, presumably due to differences in laser intensity at various axial distances from the focus position. On this point, our findings are in contrast with results reported in other well known papers, and indicate different fading at various distances from the laser focus position. The correction procedure can and should be established for the same specimen, but on a different part of the specimen from that used in the actual biological study. Calibration can thus be done on an unknown or inhomogenous object. For a series of confocal xy-scans through the immunostained cells, a corrected summation image representing total FITC-fluorescence related to UDG was obtained. Both noise removal and fading corrections were performed on each image in the series before the summation image was made. Estimates of total amounts of UDG localized in the cells and nuclei, respectively, could then be obtained. Measurement of the total cellular UDG-content by flow cytometry was also performed in order to make a comparison of the two methods for quantitative analysis. For both methods a range of approximately 4.5 was obtained between total UDG-content of cells at the 5 and 95 percentage points.

DNA Glycosylases↗
PubMed1997

Vagal afferent innervation of the atria of the rat heart reconstructed with confocal microscopy.

We have used confocal microscopy to analyze the vagal afferent innervation of the rat heart. Afferents were labeled by injecting 1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate (DiI) into the nodose ganglia of animals with prior supranodose de-efferentations, autonomic ganglia were stained with Fluoro-gold, and tissues were examined in whole mounts. Distinctively different fiber specializations were observed in the epi-, myo-, and endocardium: Afferents to the epicardium formed complexes associated with cardiac ganglia. These ganglia consisted of four major ganglionated plexuses, two on each atrium, at junctions of the major vessels with the atria. Ganglionic locations and sizes (left > right) were consistent across animals. In addition to principal neurons (PNs), significant numbers of small intensely fluorescent (SIF) cells were located in each of these plexuses, and vagal afferents provided dense pericellular varicose endings around the SIF cells in each ganglionic plexus, with few if any terminations on PNs. In the myocardium, vagal afferents formed close contacts with cardiac muscles, including conduction fibers. In the endocardium, vagal fibers formed "flower-spray" and "end-net" terminals in connective tissue. With three-dimensional reconstruction of confocal optical sections, a novel polymorphism was seen: Some fibers had one or more collaterals ending as endocardial flower sprays and other collaterals ending as myocardial intramuscular endings. Some unipolar or pseudounipolar neurons within each cardiac ganglionic plexus were retrogradely labeled from the nodose ganglia. In conclusion, vagal afferents form a heterogeneity of differentiated endings in the heart, including structured elements which may mediate chemoreceptor function, stretch reception, and local cardiac reflexes.

Afferent Pathways↗
PubMed2002

Confocal microscopy in multiple myeloma crystalline keratopathy.

PURPOSE: To report confocal microscopy findings of a patient with multiple myeloma crystalline keratopathy and the response to treatment. METHODS: Confocal microscopy images of the cornea were taken OU and the corneal crystals analyzed using the Cell Counter software. RESULTS: Numerous hyperreflective globules 6-11 nm in size were located within the corneal epithelium and anterior stroma. These crystals obscured normal architectural detail of the cornea. After 6 months of chemotherapy, confocal microscopy was repeated and demonstrated decrease in the size and number of hyperreflective globules. CONCLUSION: Confocal microscopy can enable the clinician to monitor the clinical response of multiple myeloma crystalline keratopathy to chemotherapeutic agents.

Aged↗